Systems and methods for calibrating and focusing an imaging apparatus

Abstract

Described are systems and methods to focus an imaging apparatus, in particular to autofocus an imaging apparatus in order to detect a biologic on a surface in an automated fashion. The present disclosure further provides systems and methods for calibrating an imaging apparatus by utilizing an autofocus system of an instrument in order to detect a biologic on a surface in an automated fashion.

Description

WSGR Docket No. 64100-742.601SYSTEMS AND METHODS FOR CALIBRATING AND FOCUSING AN IMAGING APPARATUS

[0001] The present application claims the benefit of United States Provisional Patent Application Number 63 / 703,524, filed on October 4, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] The use of open channels in a fluidic system can enable technical advantages as compared with closed-channel fluidic systems, for example, by enhancing functionality and accessibility for liquid handling and compatibility with consumables. Open channel fluidic systems can findutility in biomedical applications, for example, cell culture applications, hydrogel preparations, metabolite extraction and DNA sequencing, and the like. The robust performance of an open fluidic system can be useful for improved precise and reliable control of the fluidics in open channels or wells and imaging of the contents of the wells.

[0003] Challenges associated with open channel format fluidic systems may include accurately focusing an imaging apparatus on a surface of a well, for example, focusing each well across a multi-well plate open channel format fluidic system and detecting the contents of each well. This challenge can be due to variations in manufacturing tolerances and surface flatness between one or more surfaces of a multi-well plate. Deviations in surface flatness, e.g., between wells with respect to a static position of an imaging apparatus, can degrade the otherwise diffraction limited imaging resolution of the imaging apparatus thereby limiting the usefulness of the open channel fluidic system configuration.

[0004] Open-channel microfluidics are attractive for simplicity, accessibility, and low-cost fabrication, but because fluids are exposed to air, they may require human monitoring (for example, pipetting, visual checks, timed refills, contamination checks, and the like) to manage evaporation, meniscus position, reagent volume and cross-contamination.SUMMARY

[0005] Additional aspects and advantages of the present disclosure will become readily apparent from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0006] In an aspect, the present disclosure provides system for calibrating instrument imaging apparatuses, the systems comprising one or more processors and a memory storing one or more programs for execution by the one or more processors, the one or more programs comprisingWSGR Docket No. 64100-742.601 instructions to: (i) direct a first light beam from a first light source of a first imaging apparatus to a portion of a surface positioned proximate to a plurality of positions on the surface, wherein a distance between the surface and the first imaging apparatus can change by translating an objective over the plurality of positions thereby generating a plurality of objective positions; (ii) receive a reflected portion of the first light beam onto an imaging sensor of the first imaging apparatus to form an image at each position of the plurality of objective positions; (iii) calculate a centroid sensor coordinate and size metric of the reflected portion of the first light beam for each position of the plurality of objective positions identified within the image of the first imaging apparatus; (iv) fit a linear portion of a relationship between the centroid sensor coordinates of the image from the first imaging apparatus over the plurality of positions and an objective translator position for a subset of the plurality of positions; (v) direct a second light beam from a second light source to a field of view of a second imaging apparatus and receive a reflected portion of the second light beam onto at least one imaging sensor of the second imaging apparatus to f orm a second image at each position of the plurality of positions, thereby generating a second set of images from a second set of imaging sensors of the second imaging apparatus over the subset of the positions of the plurality of positions by translating the objective on a z axis; (vi) compute a focus metric from the second set of images from the second imaging apparatus over the subset of the positions of the plurality of positions; and (vii) generate a focus model based at least on an optimized focus metric from the second imaging apparatus and the reflected beam centroid sensor coordinates from the first imaging apparatus, thereby calibrating the imaging apparatuses. In some embodiments, the instructions further comprise autofocusing an instrument after calibrating the instrument imaging apparatuses, wherein autofocusing comprises: (viii) translating the surface in one or more of anx- axis and a-y axis to a different position of the surface, and (ix) translating the objective to a position to receive a second reflected portion of the first light beam onto an imaging sensor of the first imaging apparatus, wherein centroid sensor coordinates of the second reflected portion of the first light beam match a previously optimized centroid sensor coordinate from the first imaging apparatus. In some embodiments, the first light beam is non -fluorescent and the second lightbeam is fluorescent. In some embodiments, the surface comprises a first surface and is located on a bottom of a multi-welled plate, and wherein the instructions further comprise: (a) performing (i) - (vii) for a position on a second surface, wherein the second surface comprises a bottom of a well of the multi-welled plate; (b) performing (i) - (vii) for a position on a third surface, wherein the third surface comprises a top of a fluid of a fluid / air interface located above the second surface; and (c) updating a linear portion of the second surface. In some embodiments, the first surface is between about 0.1 millimeters and about 1.5 millimeters below the second surface, and wherein the third surface is about 2 millimeters to about 10 millimeters above the second surface. In someWSGR Docket No. 64100-742.601 embodiments, the position of the third surface is changeable relative to the position of the first surface or the position of the second surface, and wherein the difference between the second surface and the third surface indicates a level of the fluid in the well of the multi-welled plate. In some embodiments, fitting the linear portion of the relationship comprises: (a) using a function to determine a size metric for each image captured at each position of the plurality of objective positions corresponding to each centroid; (b) selecting the size metric which maximizes or minimizes the function; and (c) selecting a subset of centroids corresponding to (i) the selected size metric and (ii) size metrics between a first position and a second position of the plurality of positions, wherein each size metric is associated with a spot size of each image, and wherein the function comprises an exponential or parabolic function of the spot sizes of the subset of the centroids. In some embodiments, optimizing the focus metric comprises: (a) using a function to determine a focus metric for each image captured at each position of the plurality of objective positions corresponding to each centroid of the linear portion of the relationship; and (b) selecting the focus metric which maximizes or minimizes the function, wherein each focus metric is associated with a spot size of each image, and wherein the function comprises (i) a full width at half maximum (FWHM) function, (ii) a Brenner gradient function, or (iii) a Shannon Entropy of Laplacian Pyramid (SLaP) function. In some embodiments, the instructions further comprise determining (i) a local tilt, (ii) a global tilt, or (iii) a combination of (i) and (ii), of the surface from a plane of the imaging apparatuses. In some embodiments, determining the local tilt comprises: (a) obtaining a centroid for each of the plurality of positions of the surface; (b) fitting a planar surface to the plurality of positions; and (c) determining a slope of the planar surface thereby determining the local tilt. In some embodiments, determining the global tilt comprises: (a) obtaining a centroid for each of the plurality of positions of the surface; (b) fitting a parametric function to the plurality of positions of the surface; and (c) determining a slope of the parametric function thereby determining the global tilt. In some embodiments, the instructions are performed in a time of about 500 milliseconds, about 400 milliseconds, about 300 milliseconds, about 200 milliseconds, about 100 milliseconds, or less. In some embodiments, the instructions comprise using a stage to position the surface for the imaging apparatuses. In some embodiments, the instructions comprise positioning the objective in a z axis relative to the surface for the imaging apparatuses. In some embodiments, the stage is configured with a travel range between the plurality of positions of up to about 6 millimeters. In some embodiments, the stage is configured with a capture range of the plurality of positions of up to about 2 millimeters by the first imaging apparatus, the second imaging apparatus, or a combination thereof. In some embodiments, the stage is configured with a depth of focus of the plurality of positions comprising a range of up to about 5 micrometers with an accuracy of about 10% of the depth of focus from the first imageWSGR Docket No. 64100-742.601 apparatus, the second imaging apparatus, or a combination thereof. In some embodiments, a distance between a first position and a second position of the plurality of positions is from about 5 nanometers to about 5 millimeters. In some embodiments, a distance between a first position and a second position of the plurality of positions is from about 100 nanometers to about 1 millimeter. In some embodiments, a distance between a first position and a second position of the plurality of positions is approximately 40 micrometers. In some embodiments, the instructions further comprise (i) moving the first imaging apparatus, the second imaging apparatus, or a combination thereof continuously between each position of the plurality of positions at a rate, (ii) moving the imaging apparatus periodically between each position of the plurality of positions, or (iii) a combination of (i) and (ii). In some embodiments, the instructions further comprise using the calibrated image apparatuses to identify one or more particles. In some embodiments, the one or more particles comprise amplification products hybridized to fluorescently labeled polynucleotides. In some embodiments, the amplification products are immobilized on the second surface. In some embodiments, the second surf ace is the bottom of the well of the multi -welled plate selected from a 48-well multi-welled plate, a 96-well multi-welled plate, a 384-well multiwelled plate, or a 1,536-well multi-welled plate. In some embodiments, the bottom of the well comprises a (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a v shape, (v) a geometric shape, or (vi) any combination of (i) - (v). In some embodiments, the bottom of the well comprises a surface area of about 0.02 square centimeters (cm2), of about 0.03 cm2, of about 0.05 cm2, of about 0.08 cm2, of about 0.1 cm2, of about 0.2 cm2, of about 0.3 cm2, of about 0.4 cm2, of about 0.5 cm2, of about 0.6 cm2, of about 0.7 cm2, of about 0.8 cm2, of about 0.9 cm2, of about 1.0 cm2, or greater.

[0007] In an aspect, the present disclosure provides methods for calibrating imaging apparatuses, the method comprising: (a) directing a first light beam from a first light source of a first imaging apparatus to a position of a plurality of positions on a second surface; (b) receiving a reflected portion of the first light beam onto an imaging sensor of the first imaging apparatus to form a captured image for each position of the plurality of positions; (c) fitting a linear portion of a relationship between a centroid coordinate of the captured images from the first imaging apparatus at each position of the plurality of positions and an objective translator position for a subset of the plurality of positions; (d) directing a second light beam from a second light source to a field of view for a second imaging apparatus and receiving a signal from reflected light of the second light beam onto at least one imaging sensor of the second imaging apparatus to form a second captured image at each position of the plurality of positions; (e) generating the second captured image from a second set of imaging sensors of the second imaging apparatus over the subset of the plurality of positions by translating an objective on a z axis; (f) determining a focus metric from a set ofWSGR Docket No. 64100-742.601 captured images from the second imaging apparatus over the subset of the plurality of positions; and (g) generating a focus model based at least on an optimized focus metric from the second imaging apparatus and reflected beam centroid sensor coordinates from the first imaging apparatus, thereby calibrating the first imaging apparatus and the second imaging apparatus of an instrument. In some embodiments, the method further comprises autofocusing an instrument after calibrating the instrument imaging apparatuses, wherein the autofocusing comprises: (h) translating the second surface in one or more of an x-axis or a y-axis to a different position of the second surface, and (i) translating the objective to a position to receive a reflected portion of the light beam onto an imaging sensor of the first imaging apparatus, wherein the centroid sensor coordinates of the reflected portion of the lightbeam match a previously optimized centroid sensor coordinate. In some embodiments, the first light beam of the first imaging apparatus is non- fluorescent and the second light beam of the second imaging apparatus is fluorescent. In some embodiments, the method further comprises repeating (a) - (d) for each of one or more other positions of the second surface. In some embodiments, the method further comprises (i) performing (a) - (d) for a portion of a first surface, wherein the first surface is below the second surface; (ii) performing (a) - (d) for a portion of a third surface, wherein the third surface is above the second surface; and (iii) updating the linear portion of the second surface. In some embodiments, the first surface is provided on abottom of amulti -welled plate, wherein the second surface is provided on a bottom of a well of a multi -welled plate, and wherein the third surface is at a fluid / air interface provided in the well above the second surface. In some embodiments, the first surface is about 0.1 millimeters to about 1.5 millimeters below the second surface, and wherein the third surface is about 2 millimeters to about 10 millimeters above the second surface. In some embodiments, a difference between the second surface and the third surface indicates a level of fluid in the well of the multi -welled plate. In some embodiments, fitting the linear portion of the relationship comprises: (i) using a function to determine a size metric for each image corresponding to each centroid captured at each position of the plurality of positions; (i) selecting the size metric which maximizes or minimizes the function; and (i) selecting a subset of the centroids captured at each position of the plurality of positions corresponding to (A) the selected size metric and (B) size metrics determined between the first position and the second position of the plurality of positions, wherein each size metric is associated with a spot size of each captured image, and wherein the function comprises an exponential or parabolic function of combined spot sizes of the subset of the centroids. In some embodiments, optimizing the focus metric comprises using a function to determine the focus metric, wherein the function comprises (i) a full width at half maximum (FWHM) function, (ii) a Brenner gradient function, or (iii) a Shannon Entropy of Laplacian Pyramid (SLaP) function. In some embodiments, the method further comprisesWSGR Docket No. 64100-742.601 determining (A) a local tilt, (B) a global tilt, or (C) a combination of (A) and (B), of the second surface from a plane of as first imaging apparatus, a second imaging apparatus, or a combination thereof. In some embodiments, determining the local tilt comprises: (a) obtaining a centroid for each of the plurality of positions of the second surface; (b) fitting a planar surface to the plurality of positions; and (c) determining a slope of the planar surface thereby determining the local tilt. In some embodiments, determining the global tilt comprises: (a) obtaining a centroid for each of the plurality of positions of the second surface; (b) fitting a parametric surface to the plurality of positions of the surface; and (c) determining a slope of the parametric surf acethereby determining the global tilt. In some embodiments, the method further comprises using a stage to position the plurality of positions with respect to the first imaging apparatus, the second imaging apparatus, or a combination thereof. In some embodiments, the method further comprises positioning the objective in a z axis relative to the second surface for the first imaging apparatus, the second imaging apparatus, or a combination thereof. In some embodiments, the stage is configured with a travel range of the plurality of positions of up to about 6 millimeters from the first image apparatus, the second imaging apparatus, or a combination thereof. In some embodiments, the stage is configured with a capture range of the plurality of positions of up to about 2 millimeters from the first image apparatus, the second imaging apparatus, or a combination thereof. In some embodiments, the stage is configured with a depth of focus of the plurality of positions of up to about 5 micrometers with an accuracy of about 10% of the depth of focus from the first image apparatus, the second imaging apparatus, or a combination thereof. In some embodiments, a distance between a first position and a second position of the plurality of positions comprises about 100 micrometers (pm), about 90 pm, about 80 pm, about 70 pm, about 60 pm, about 50 pm, about 40 pm, about 30 pm, about 20 pm, or about 10 pm. In some embodiments, the method further comprises (i) moving the first imaging apparatus, the second imaging apparatus, or a combination thereof continuously between each position of the plurality of positions at a rate, (ii) moving the first imaging apparatus, the second imaging apparatus, or a combination thereof periodically between each position of the plurality of positions, or (iii) a combination of (i) and (ii). In some embodiments, the method further comprises usingthe first calibrated image apparatus and the second calibrated image apparatus to identify one or more particles immobilized on the second surface. In some embodiments, the one or more particles comprise amplification products hybridized to fluorescently labeled polynucleotides. In some embodiments, the second surface comprises one or more other surface positions of a well of a 48-well multi-welled plate, a 96-well multi-welled plate, a 384-well multi-welled plate, or a 1536-well multi-welled plate. In some embodiments, a bottom of the well comprises (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a v-shape, (v) a geometric shape, or (vi) any combination of (i) - (v). InWSGR Docket No. 64100-742.601 some embodiments, the bottom of the well comprises a surface area of about 0.02 square centimeters (cm2), of about 0.03 cm2, of about 0.05 cm2, of about 0.08 cm2, of about 0.1 cm2, of about 0.2 cm2, of about 0.3 cm2, of about 0.4 cm2, of about 0.5 cm2, of about 0.6 cm2, of about 0.7 cm2, of about 0.8 cm2, of about 0.9 cm2, of about 1.0 cm2, or greater.

[0008] In an aspect, the present disclosure provides methods for determining a fluid level in a well, the method comprising: (a) directing a light beam from alight source of a calibrated imaging apparatus to a position on a second surface and a position on a third surface of a well positioned proximate to a plurality of positions from a sensor, wherein a fluid is disposed in the well above the second surface, and wherein a fluid / air interface in the well is the third surface; (b) receiving a portion of a reflected light beam from the second surface and a portion of the reflected light beam from the third surface onto the sensor at each position of the plurality of positions of the second surface and at each position of the plurality of positions of the third surface; (c) using the portion of the reflected light beam from the second surface and the portion of the reflected light beam from the third surface to determine a position of the second surface and a position of the third surface based on the plurality of positions; and (d) determining the fluid level in the well from the determined position of the second surface and the determined position of the third surface. In some embodiments, the second surface is a bottom of a well of a multi -welled plate. In some embodiments, the third surface is about 2 millimeters to about 10 millimeters above the second surface. In some embodiments, the second surface and the third surface are variable in position relative to each other. In some embodiments, the method further comprises determining (i) a local tilt, (ii) a global tilt, or (iii) a combination of (i) and (ii), of the second surface from a plane of the sensor. In some embodiments, determining the local tilt comprises: (a) obtaining a centroid of an image of the reflected portion of the light beam for each of the plurality of positions of the second surface; (b) fitting a planar surface to the plurality of positions; and (c) determining a slope of the planar surface thereby determining the local tilt. In some embodiments, determining the global tilt comprises: (a) obtaining a centroid of an image of the reflected portion of the light beam for each of the plurality of positions of the second surface; (b) fitting a parametric surface to the plurality of positions of the second surface; and (c) determining a slope of the parametric surface thereby determining the global tilt. In some embodiments, the method further comprises using a stage to position the sensor at the plurality of positions. In some embodiments, the method further comprises positioning an obj ective of the calibrated imaging apparatusesin a z axis relative to the surface. In some embodiments, the stage is configured with a travel range of the plurality of positions from the sensor of up to about 6 millimeters. In some embodiments, the stage is configured with a capture range of the plurality of positions from the sensor of a range up to about 2 millimeters. In some embodiments, the stage is configured with a depth of focus of the pluralityWSGR Docket No. 64100-742.601 of positions from the sensor of a range of up to about 5 micrometers with an accuracy of about 10% of the depth of focus. In some embodiments, a distance between a first position and an additional position of the plurality of positions on the second surface comprises about 100 micrometers (pm), about 90 pm, about 80 pm, about 70 pm, about 60 pm, about 50 pm, about 40 pm, about 30 pm, about 20 pm, or about 10 pm. In some embodiments, the method further comprises (i) moving the sensor continuously between each position of the plurality of positions at a rate, (ii) moving the sensor periodically between each position of the plurality of positions, or (iii) a combination of (i) and (ii). In some embodiments, the method further comprises focusing the sensor on one or more particles immobilized on the second surface. In some embodiments, the one or more particles comprise amplification products hybridized to fluorescently labeled polynucleotides. In some embodiments, the second surface is the bottom of a well of a 48-well multi-welled plate, a 96-well multi-welled plate, a 384-well multi-welled plate, or a 1536-well multi-welled plate. In some embodiments, the bottom of the well comprises (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a geometric shape, or (v) any combination of (1) - (iv). In some embodiments, thebottom of the well comprises a surface area of about 0.02 square centimeters (cm2), of about 0.03 cm2, of about 0.05 cm2, of about 0.08 cm2, of about 0.1 cm2, of about 0.2 cm2, of about 0.3 cm2, of about 0.4 cm2, of about 0.5 cm2, of about 0.6 cm2, of about 0.7 cm2, of about 0.8 cm2, of about 0.9 cm2, of about 1.0 cm2, or greater. In some embodiments, the determined fluid level in the well is used to detect a fluidic failure of a fluidic system.

[0009] In an aspect, the present disclosure provides imaging apparatuses, the apparatus comprising: (a) a light source configured to direct a light beam to a portion of a surface positioned proximate to a plurality of positions relative to an imaging apparatus; (b) a sensor configured to receive a reflected portion of the light beam onto the sensor of the imaging apparatus thereby forming an image at each position of the plurality of positions; and (c) an automatic focuser configured to: (i) fit a linear portion of a relationship to a centroid of each image between a first position and a second position of the plurality of positions; (ii) optimize a focus metric obtained from the linear portion of the relationship; and (iii) generate a focus model based at least on the optimized focus metric.INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict theWSGR Docket No. 64100-742.601 present disclosure contained in the specification, the specification is intended to supersede or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying.

[0012] FIG. 1 shows an example drawing of a component device of a self-calib ration system of the present disclosure, as described in some embodiments herein .

[0013] FIG. 2 shows an example schematic illustrating the orientation of the self-calib ration system imaging apparatus with respect to an exemplary 96 well plate, as described in some embodiments herein.

[0014] FIG. 3A shows an example schematic of the translational range of the imaging apparatus with respect to a tilted surface, as described in some embodiments herein.

[0015] FIG.3B shows an example schematic of the translational range of the imaging apparatus with respect to a de-tiled surface, as described in some embodiments herein.

[0016] FIG. 4 shows an example flow diagram for an autofocus method of receiving light reflected by one or more interfaces with an imaging apparatus and determining a focal correction for the imaging apparatus, as described in some embodiments herein.

[0017] FIG. 5A shows an example schematic diagram of a path of a beam emitted by the imaging apparatus directed towards one or more surfaces, as described in some embodiments herein.

[0018] FIG. 5B shows an example of a corresponding reflected one or more beams of light collected or detected by an imaging apparatus as the imaging apparatus is translated towards a surface of the one or more surfaces, as described in some embodiments herein .

[0019] FIG. 6A shows an example graph of a position of a reflectedbeam of a surface imaged on a detector of an imaging apparatus as the imaging apparatus is translated, as described in some embodiments herein.

[0020] FIG. 6B shows an example measured spot size of the reflected beam off of the surfaced imaged on the detector of the imaging apparatus as the imaging apparatus is translated, as described in some embodiments herein.

[0021] FIG. 7A shows an exemplary graph of a detected size of a beam reflected off of a surface detected or collected by a translating imaging apparatus and a correspondingidentified focal range of the imaging apparatus, as described in some embodiments herein.WSGR Docket No. 64100-742.601

[0022] FIG. 7B shows an example corresponding position of a detected reflected beam on a detector of the imaging apparatus within the identified focal range of the imaging apparatus determined from the graph of the detected size of the beam, as described in some embodiments herein.

[0023] FIG.7C shows an example of the full-width half max of an imaged signal of the biological sample within the identified focal range, as described in some embodiments herein .

[0024] FIG. 8A shows an example variability in detected beam spot position on a detector of the imaging apparatus within an identified focal range of the imaging apparatus across one or more portions of a surface, as described in some embodiments herein.

[0025] FIG. 8B shows an example corresponding adjustment in a position of the imaging apparatus with respectto one or more portions of the surface to bringthe imaging apparatus within the optimized focal range to image the one or more portions of the surface, as described in some embodiments herein.

[0026] FIG. 9A shows one or more example approaches in determining a local tilt using wells of a full plate, within one well of a plate, and between several wells of a plate, as describedin some embodiments herein.

[0027] FIG. 9B shows an example graph of centroid positions of a reflected beam off of a well surface for a plurality of positions within the well, as described in some embodiments herein.

[0028] FIG. 9C shows an example three-dimensional graph of a plurality of positions of a well surface used to generate a plane to correct for local tilt, as describedin some embodiments herein.

[0029] FIG. 9D shows example configurations or spatial pattern of the plurality of positions on a well surface that are imaged to determine a local de-tilt of a well surface, as described in some embodiments herein.

[0030] FIG. 10 shows an example software flow diagram for active tip -tilt method, as described in some embodiments herein.

[0031] FIG. 11 shows an example flow diagram for a method of calibrating an imaging instrument, as described in some embodiments herein.

[0032] FIG. 12A shows an example flow diagram for a method of measuring a curvature of one or more surfaces of a well plate, as described in some embodiments herein.

[0033] FIG. 12B shows an example quadratic model fit to the one or more measurements of curvature, as described in some embodiments herein.

[0034] FIG. 13A shows an example flow diagram for a method of validating a tip-tilt encoder reading, as described in some embodiments herein.

[0035] FIG. 13B shows an example plot of encoder readings over time, as described in some embodiments herein.WSGR Docket No. 64100-742.601

[0036] FIG. 14 shows an example flow diagram for a method of verifying encoder tip -tilt values match values of tip and tilt of the surface sent to the one or more tip -tilt motors, as described in some embodiments herein.

[0037] FIG. 15 shows an example flow diagram for a triangulation method of determining a tilt of a well plate and actuating one or more motors to tip and tilt the well plate to correct for the tilt of the well plate, as described in some embodiments herein.

[0038] FIG. 16 shows an example flow diagram for a method of autofocusing on a surface of a well, as described in some embodiments herein.

[0039] FIG. 17 shows an example flow diagram for a method of determining a start and stop position of a surface from a start and stop centroid of a beam of light reflected from the surface, as described in some embodiments herein.

[0040] FIG. 18 shows an example flow diagram for a method of determining a start and end z position of a linear range of focus on a surface and a z position corresponding to the minimum beam waist width of a reflected beam within the linear range of focus, as described in some embodiments herein.

[0041] FIG. 19 shows an example flow diagram fora method of generating a focus model for one or more light sources, as described in some embodiments herein.

[0042] FIG. 20 shows an example flow diagram for a method of testing a generated focus model, as described in some embodiments herein.

[0043] FIG. 21 shows an example flow diagram for a method of determining a translation of an imaging apparatus to focus the imaging apparatus on a surface of a well, as described in some embodiments herein.

[0044] FIG. 22 shows an example flow diagram for a method of calibrating an imaging system, as described in some embodiments herein.

[0045] FIG. 23 shows an example flow diagram for a method of determining a fluid level in a well, as described in some embodiments herein.

[0046] FIG. 24 shows an example schematic of a computer system configured to implement the methods described elsewhere, as described in some embodiments herein.

[0047] FIG. 25 shows an example flow diagram of processing acquired centroid image data of one or more light beams reflected off of one or more surfaces of a well plate to identify a corresponding surface in connection with the detected reflected light beam(s), as described in some embodiments herein.

[0048] FIG. 26 shows an example plot of a position of one or more detected light beams reflected off of one or more surfaces and corresponding linear fit and error bars, as described in some embodiments herein.WSGR Docket No. 64100-742.601

[0049] FIG. 27A shows an example of interface image spot classification using an engineering margin of 32, without correction by RANSAC regressor algorithm.

[0050] FIG. 27B shown an example of interface image spot classification using an engineering margin of 16 without correction by RANSAC regressor algorithm.

[0051] FIG. 28 shows an example of interface spot image classification using an engineering margin of 16 with correction by RANSAC regressor algorithm.

[0052] FIG. 29 shows an example of the RANSAC algorithm that can be implemented for spot image classification.

[0053] FIG. 30 shows an example workflow for measuring dispensing of reagents into a well, in this example a wash run workflow.

[0054] FIG. 31 shows an example of implementing an autofocus operation in the workflow of FIG. 30

[0055] FIG. 32A shows an example graph for calculating 9 x and 9 y values, or the plate-tilt with respect to the aspirator nozzle, for 9 x as 9 y changes.

[0056] FIG.32B shows an example graph for calculating 9 x and 9 y values, or the plate-tilt with respect to the aspirator nozzle, for 9 y and 9 x changes.

[0057] FIG. 33 shows an example graph of aspiration target volumes across different columns of a well plate pre and post calibration.

[0058] FIG. 34 shows an example graph of wash and dehybridization reagent dispensed volumes pre and post calibration.DETAILED DESCRIPTION

[0059] There are challenges associated with open channel format fluidic systems. For example, challenges may include accurately focusing an imaging apparatus on a surface of a well by focusing each well across a multi -well plate open channel format fluidic system and detecting the contents of each well. As such, there exists a need for systems and methods configured to compensate for surface non-uniformity of one or more surfaces of a multi-well plate and to autofocus an imaging apparatus on a surface of the one or more wells. There is a need for systems and methods that are configured to precisely and rapidly measure a fluid level, for example, by measuring a fluid level within each well of a multi -well plate, to automate and leverage the benefits of open fluidic systems.

[0060] Autofocus in fluorescence microscopy systems can work by utilizing a software -based image analysis method or a hardware-based optical method to determine the degree of defocus and adjust the focus accordingly.WSGR Docket No. 64100-742.601

[0061] For software-based image analysis autofocus, acquired microscope images can be analyzed to calculate a focus metric that quantifies the sharpness or contrast of the image. The system can then adjustthe focus by movingthe sample orthe objective lens to maximize the focus metric. An important aspect of the software-based image analysis autofocus method include analyzing normal microscope images to determine defocus that can integrate seamlessly into a pre-existing image acquisition and processing workflow. However, such autofocusing methods are limited in the thickness of samples that they may image within their depth of focus. Moreover, the software-based image analysis autofocus methods may also (i) interrupt normal image acquisition to refocus and (ii) increase light exposure to a sample that may result in additional imaging time and risk of damaging the sample by phototoxicity, respectively.

[0062] For hardware-based optical autofocus, a dedicated optical system, which can be separate from the main microscope optics, may be utilized to measure the distance to the sample and determine the defocus. Important aspects of the hardware -based optical autofocus methods can include: (i) using a near-infrared (NIR) laser beam focused on the sample, (ii) measuring the reflected NIR beam to calculate defocus, (iii) performing autofocusing operation in parallel with normal image acquisition, (iv) minimizing phototoxicity to the sample, (v) focusing for extended 3D samples beyond the depth of focus, and (vi) using multiple focus detection point for improved stability.

[0063] When used alone, software and hardware-based methods have some utility in different fluorescence microscopy applications based on the specific sample requirements and imaging modalities used, e.g., time lapse microscopy or single molecule localization microscopy. However, when used alone, such methods can be technically deficient for at least reasons described elsewhere herein.

[0064] The present disclosure provides an improved technical approach over the software and hardware methods for focusing on one or more surfaces of an open fluidic system platform. For example, systems and methods herein can provide at least a technical solution for combining both software and hardware elements into a single workflow. Improvements may include at least allowing for automatic focusing or autofocusing of an imaging apparatus optics on a large format well plate to address shortcomings of autofocusing solutions. For example, systems and methods herein can provide atleast a technical solution for maintainingfocus across afield of view(FOV), e.g., a FOV of about 3 mm x about 3 mm, which can be considered widefield or a large FOV for autofocus in view of the imaging range of microscopy systems at up to about 1 mm x about 1mm.

[0065] In some embodiments, an optical system provides a readout capability of the instrument. An optical system useful in calibrating and autofocusing an instrument as described herein can comprise several sub-systems, including but not limited to a light engine and an optical module,WSGR Docket No. 64100-742.601 both of which can be used in tandem to image a surface. In some embodiments, the light engine supports one or more different light sources through separate fibers coupled to the optical module. In some embodiments, the one or more different light sources can be activated independently by the instrument control software. In some embodiments, an optical module sub-system comprises of one or more imaging apparatuses, each of which can comprise several additional optical components, including but not limited to mirrors, filters, sensors, stages, and beam shapers. In some embodiments, an optical module comprises an objective that is mounted on a translator. A translator enables the objective to move up and down relative to the imaging apparatuses. In some embodiments, an objective is proximal to, but not directly coupled to, each of the imaging apparatuses.

[0066] In some embodiments, a first imaging apparatus can be coupled to an optical fiber from a light engine. A first imaging apparatus can direct a light beam from the light source through various optical elements and through an objective of the optical module to a portion of a surface positioned proximate to an imaging apparatus. This first imaging apparatus can further receive a reflected beam of light from the surface back through the objective, optical elements, and on an imaging sensor located within the first imaging apparatus. A reflected beam of light can be received by an imaging detector of the first imaging apparatus thereby forming an image. The image can be transferred from the optical module to a computer system capable of analyzing the image. For example, a computer system that receives an image from the optical module can compute the centroid of the image and determine descriptive statistics of the identified image, for example, image size, circularity, intensity, signal-to-noise ratio, and the like. If the objective of the optical module is translated over a plurality of positions, the centroid of the reflected portion of the light beam on the first imaging apparatus sensor is able to shift, thereby providing information on, for example, the distance between a surface and the objective.

[0067] In some embodiments, a second imaging apparatus is coupled to a second optical fiber from a light engine. A second imaging apparatus can direct a light beam from the light source through various optical elements and through the objective to a portion of a surface positioned proximate to the imaging apparatus. In some embodiments, the second imaging apparatus is further capable of receiving fluorescence signal generated at a surface back through the objective, separate optical elements, and onto a set of secondary imaging sensors located within the second imaging apparatus. The received light signal onto a set of imaging sensors of the second imaging apparatus forms an image per sensor. In some embodiments, one image per sensor is formed from received light that is received from a set of imaging sensors of a second imaging apparatus. In some embodiments, the images that are formed are transferred from the optical module onto a computer system for image analysis. In some embodiments, image analysis can compriseWSGR Docket No. 64100-742.601 identifying discrete features of interest and computing focus metrics using a series of tunable algorithms. In some embodiments, when the objective is translated over a plurality of positions, the focus metrics calculated from the received fluorescence signal on the sensors provides information on the focus quality of the images as a function of the distance between the surface and the objective.

[0068] As such the present disclosure provides for an accuracy of at least about 300 nanometer (nm) accuracy within an at least about 5 mm travel for autofocusing on a surface, e.g., a surface of a well of a well plate. In some embodiments, the autofocusing methods or systems, described herein, may be robust to both local tip-tilt, planar tip-tilt, global tip-tilt, non-linear tip-tilt, or any combination thereof. The present disclosure provides systems or methods that may determineboth local tip-tilt, planar tip-tilt, overall global tip-tilt, non-linear tip-tilt, or any combination thereof. The present disclosure further provides for systems or methods that identify and / or determine a focus position of a surface and / or one or more points across a surface and / or one or more surfaces for each autofocusing operation, described herein, within up to about 1%, up to about 2%, up to about 3%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, or up to about 15% of a difference between the focus position of the surface determined by a focus metric e.g., full-width half max, Brenner metric, Shannon entropy, or any combination thereof. For example, the precision and accuracy of identifying one or more focus positions of one or more surfaces ensures fast and accurate imaging of an entire well plate.

[0069] In some embodiments, the present disclosure provides a software and hardware integrated solution for at least about a 300 nm accuracy within an at least about 5 mm travel range for autofocusing on a surface. In some embodiments, the surface may comprise a biological sample disposed or provided thereto. The biological sample may be a spherical biological sample. A biological sample may be prokaryotic or eukaryotic in nature. Examples of a biological sample may include, but are not limited to, a tissue sample, a cell sample, a biopsy sample, an autopsy sample, a blood sample, a bodily fluid sample, a bodily solid sample, nucleic acids derived from one of more sample types, extracted DNA, extracted RNA, cDNA, cell free DNA derived from a blood, serum, a plasma sample, or any combination thereof. In some embodiments, the biological sample may comprise a human sample, a non-human sample, or any combination thereof. The present disclosure may notbe limited by the biological sample(s) associated with a surface upon which autofocusing as described herein is performed.Systems for Calibrating an Imaging Apparatus

[0070] In some cases, as illustrated in the example of FIG. 1, a system for calibrating a focus of an imaging apparatus or performing autofocusing may comprise: (a) a stage 100 coupled to aWSGR Docket No. 64100-742.601 substrate 101 comprising one or more wells, where the stage 100 rotates about a first axis (106a, 106b) with a first motor 109 and about a second axis (105a, 105b) with a second motor 108, wherein the first axis and the second axis are orthogonal; (b) an imaging apparatus 200 optically coupled to one or more surfaces (902, 904, 908) of the one or more wells (910), where the imaging apparatus 200 determine a slope of a plane on a portion of the one or more surfaces of the one or more wells; and (c) one or more processors electrically coupledto the stage and the optics module, wherein the one or more processors actuate the first motor 109 to rotate the stage about the first axis (106a, 106b) and the second motor 108 to rotate the stage 100 about the second axis (105a, 105b) based on at least the slope of the plane on the portion of the one or more surfaces of the one or more wells, thereby calibrating the focus of the imaging apparatus on the one or more surfaces of the one or more wells. In some cases, the imaging apparatus may comprise a first imaging apparatus coupled to a second imaging apparatus.

[0071] In some embodiments, systems described herein may determine the slope of an identified focal plane for one or more surfaces over one or more of regions or one or more locations across the one or more surfaces. In some embodiments, the one or more surfaces may comprise one or more surfaces of a well plate, as shown in the example of FIG. 9A. In some embodiments, the one or more regions or one or more locations may comprise a plurality of regions or a plurality of locations across non-adjacent wells of amulti-wellplate 910a. In some embodiments, the plurality of regions or the plurality of locations may comprise a plurality of regions or a plurality of locations within one well 910b, further illustrated in the example of FIG. 9D. In some embodiments, the plurality of regions or the plurality of locations may comprise a plurality of regions or a plurality of locations across a plurality of adjacent wells of a multi-well plate 910c.

[0072] In some embodiments, the stage may comprise a tip -tilt stage module, or a software module (as shown in the examples of FIGs. 10-21) that may direct the autofocusing methods, generation of focus models or focus ranges of an imaging apparatus, or a combination thereof, described herein, that leverage both optics and tip-tilt stage modules. FIG. 1 demonstrates an example tip-tilt module 100 for use in systems or methods of the present disclosure. The tip-tilt module 100 is viewed from the top down and may comprise a substrate 101, e.g., a well plate. In some embodiments, the well plate may comprise 96 wells. In some embodiments, the substrate may be a slide, a well plate with fewer wells, e.g., such as a 48 well plate, a 24 well plate, an eight well plate, a six well plate, or a well plate with more than 96 wells such as a 384 well plate, or a 1,536 well plate. In some instances, the well plate may comprise a bottom substrate coupled to a top substrate, where the top substrate may comprise one or more regions and / or sections where the top substrate material may be removed from top substrate. In some cases, the one or more sections where the top substrate material is removed may comprise circular sections, squareWSGR Docket No. 64100-742.601 sections, and / or triangular sections. In some cases, the one or more sections removed from the top substrate may produce and / or provide one or more wells within the one or more sections removed from the top substrate when the top substrate is coupled to the bottom substrate. The present disclosure may not be limited to the substrate on which autofocusing is being performed. In some embodiments, the bottom substrate and / orbottom surfaceof the well plate may comprise optically clear glass such that measurements can be performed on or through the bottom of the substrate, on top of the substrate, or above the substrate. Other substrates may include, but are not limited to a glass coverslip, a flow cell, a glass slide, an aperture target, or any combination thereof. In some embodiments, the substrate 101 may be removably affixed in a nest or receptacle 102. In some embodiments, the nest may be surrounded by a midframe 103. In some embodiments, the midframe 103 may be mounted in a fork apparatus 104. In some embodiments, the nest or receptacle 102 may be connected to a first motor 108 and a second motor 109, via the midframe 103. In some embodiments, the first motor 108 may comprise a theta X motor, and the second motor 109 may comprise a theta Y motor. In some embodiments, the one or more motors (108, 109) may rotate or tilt the substrate 101, on an X axis, a Y axis and / or a Z axis. In some embodiments, the second motor 109 and associated cam may rotate the nest or receptacle 102 with respect to the midframe 103 and may provide tilt orrotation in the Y dimension aboutthe second axis (105a, 105b). In some embodiments, the first motor 108 and associated cam may rotate the midframe 103 with respect to the fork apparatus 104 and may provide tilt or rotation in the X dimension about the first axis (106a, 106b). The arrows shown in the example of FIG. 1 demonstrate the rotation of the substrate 101 when the first motor 108 or the second motor 109 are activated or actuated for tipping, tilting, or rotating the substrate. In some embodiments, the motor controller 107 may provide a driving signal to the second motor 109 and the first motor 108 to tip, tilt, or rotate the second motor 109 or the first motor 108, as directed by the software, described herein.

[0073] As shown in the example of FIG. 3A, in some embodiments, when the slope of a surface of a well 302 with respectto an optical axis of a light source emission 304 (with field of view 303) of the one or more light sources emitted by an objective lens 202 is determined, the objective lens may translate a distance 305 away from a bottom surface of the well 302 to prevent the bottom of the well to contact or collide with the objective lens 202 when the well is tip, tilted, or rotated, as shown in the example of FIG. 3B. In some cases, the distance 305 may comprise at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, at least 10 mm, at least 11 mm, or at least 12 mm. In some embodiments, the distance may be determined from methods of determining a focus model or an identified focus range of one or more surfaces, as described herein. A side cross-sectional view of the tip-tilt module 100,WSGR Docket No. 64100-742.601 midframe 103, nest or receptacle 102 is shown in the example of FIG. 2 to reveal the substrate 101 disposed or provided superior to the imaging apparatus 200.

[0074] In some embodiments, an imaging apparatus 200 may comprise an optical imaging device 201 comprising one or more light sources and one or more detectors for providing light to the substrate 101 and detecting reflected light back from the substrate 101, respectively. In some cases, the imaging apparatus 200 may be coupledto a second imaging apparatus, where the second imaging apparatus may be optically coupled to one or more surfaces of a well plate with one or more biological samples disposed thereto. In some cases, the imaging apparatus 200 may determine an optical focus of the second imaging apparatus where the second imaging apparatus images the one or more biological samples disposed on the one or more surfaces and / or one or more regions of the well plate, described herein. In some cases, the optical focus may comprise a distance (e.g., a z-position) of the imaging apparatus coupled to the second imaging apparatus from one or more surfaces of a well plate. In some cases, the optical focus of the second imaging apparatus may comprisethe optical focus of the second imagingapparatusacross all fields of view of imaging the one or more surfaces and / or one or more regions of a well plate. In some cases, the imaging apparatus 200 and the second imaging apparatus may be coupledto a z-stage, described herein. In some cases, the z-stage may translate and / or move the imaging apparatus and / or the imaging apparatus coupled to the second imaging apparatus towards and / or away from the one or more surfaces of a well plate. In some embodiments, the one or more detectors of the imaging apparatus may comprise one or more sensors. In some embodiments, the objective lens 202 of the imaging apparatus 200 may be inverted and inferior to the tip-tilt module 100. In some embodiments, the objective lens 202 of the imaging apparatus 200 may disposed at a distance from the tip-tilt module 100 or a surface of the substrate 101 (e.g., a well of a multi-well plate). In some embodiments, light from the optical imaging device 201 may be directed or deliver up through the objective lens 202 to one or more surfaces of the substrate 101. In some embodiments, reflected light incident on the one or more surfaces of the substrate may travel back from the substrate 101 through the objective lens 202 to the detector in the imaging apparatus 200. In some embodiments, the one or more light sources of the imaging apparatus may include one or more LED lights or one or more lasers. In some cases, the one or more light sources may comprise one or more arrays of LED lights, one or more arrays of lasers, or a combination thereof. In some embodiments, the one or more detectors may comprise one or more sensors capable of detecting one or more fluorescence emission spectra, e.g., wavelength specific filters for detecting different wavelengths of emitted light. In some embodiments, one or more detectors may comprise a complementary metal oxide semiconductor (CMOS) sensor, one or more event-based sensors, or a combination thereof.WSGR Docket No. 64100-742.601

[0075] Further, in some embodiments, the objective lens 202 can travel through an imaging plane range 203, also referred to as the Z-travel range, when performing autofocus methods, operations, or functions, described herein. In some embodiments, the tip-tilt module 100 may move the substrate 101 in and X and Y direction, whereas the objective lens 202 may move in a Z direction, and where the Z direction is perpendicular to the X or Y direction. In some embodiments, the imaging apparatus 200 may remain static where the objective lens 202 may be capable of a Z range of motion relative to the tip-tilt module 100.

[0076] As such, when referring to FIG. 1 and FIG. 2, the autofocus software pipeline can direct the theta X motor 108 and theta Y motor 109 to tip, tilt, or rotate the substrate in an X or Y direction, or to translate the objective lens 202 in a Z direction, designed to bring the substrate 101 or a biological sample located on the substrate into focus on the one or more detectors of the imaging apparatus 200.

[0077] In some embodiments, the one or more detectors of the imaging apparatus 200 may detect a beam centroid position (as shown in the examples of FIGs. 6A 610 and 7B 740, beam size (e.g, diameter, as shown in the examples of FIGs.6B 630 and 7A 720), or a reflected beam as illustrated in the example of FIG. 5A (514, 516, 518) of the one or more light sources 500 reflected from one or more surfaces or interfaces (502, 504, 508) to determine a focus range, focus metric, or focus model to image one or more portions of the one or more surfaces or interfaces in focus with the imaging apparatus 200. In some embodiments, the diameter of the reflected beam of the one or more light sources reflected from the one or more surfaces or one or more interfaces, may be utilized to determine a center of a linear range of focus of the imaging apparatus in focusing on the one or more portions of the one or more surfaces, as shown in the examples of FIGs. 6C 620 and 7B 750. In some embodiments, the diameter of the reflected beam (as shown in the examples of FIGs. 6B 630 and 7A 720) of the one or more light sources reflected from one or more surfaces or interfaces may be used to define a range of beam centroid positions (as shown in the examples of FIGs. 6A 620 and 7B) within a linear range (e.g., an optimized focus range or focus position within a determined linear range) of focus positions of the imaging apparatus. In some embodiments, the one or more surfaces may comprise one or more surfaces of a well 510 (e.g, a multi-well plate) or interfaces 506 disposed or provided within the well, as shown in the example of FIG. 5A

[0078] In some embodiments, the focus metric, focus model, or focus range determined by systems and methods described herein, may be used to calibrate the focus of the imaging apparatus. In some embodiments, the calibration may comprise de -tilting, tilting, tipping, or rotating the surface imaged. In some embodiments, one or more portions of the surface, e.g., one or more non-adjacent wells of a multi-well plate may require varying degrees of de-tilting, tiltingWSGR Docket No. 64100-742.601 tipping, or rotation, based on the non-uniformity of the well surfaces across the multi -well plate. In some cases, after calibrating the imaging apparatus, the linear range of the depth focus model or range 840, as shown in the example of FIG. 8 A, may be used to determine a shift in a position of the imaging apparatus for the best focus at Cx 800 and the best focus of Z 805 with respect to a first position 820 and a second position 830 to identify the best focus 810. In some cases, the shift in position may be used to move the imaging apparatus to an optimized focus range, as shown in the example of FIG. 8B, for example ACx 850 and AZ 860 to obtain the final best focus 810. For example, a first well of a multi -well plate may comprise a different distance of shiftin position of the imaging apparatus than a second well of the multi -well plate to position the imaging apparatus within the focus range of the one or more surfaces. This may result from the non -planar surfaces or inherit warp of a substrate (e.g., a bottom substrate coupled to a top substrate of multiwell plate or one or more surfaces of a single body multi -well plate, described herein) of a multiwell plate across a plurality of wells of the multi-well plate.

[0079] Aspects of the disclosure provided herein describe a system for calibrating an imaging apparatus 200. The systems may comprise, as illustrated in the example of FIG. 24, one or more processors 2402 and memory (2404, 2410) storing one or more programs for execution by the one or more processors, the one or more programs comprising instructions to (i) direct one or more light sources (e.g. , a light beam) 500, as illustrated in the examples of FIG. 5 A, from a light source to a portion of a surface (502, 504, 508) positioned proximate to a plurality of positions from an imaging apparatus 200, and (ii) receive a reflected portion of the lightbeam (514, 516, 518) onto an imaging detector of the imaging apparatus to form an image at each position of the plurality of positions. The one or more program instructions further comprise (iii) fitting a linear portion of a curve to a centroid of each image between a first position and a second position of the plurality of positions, (iv) optimizing a focus metric obtained from the linear portion of the curve, and (v) generating a focus model based at least on the optimized focus metric thereby calibrating the imaging apparatus. In some embodiments, a distance between the first position and the second position of the plurality of positions may comprise a distance of at most about 100 pm, at most about 90 pm, at most about 80 pm, at most about 70 pm, at most about 60 pm, at most about 50 pm, at most about 40 pm, at most about 30 pm, at most about 20 pm, or at most about 10 pm. In some embodiments, the distance may be about40 pm. In some embodiments, the instructions may comprise (i) moving the imaging apparatus continuously at a rate between each position of the plurality of positions, (ii) moving the imaging apparatus periodically at a period between each position of the plurality of positions, or (iii) a combination of (i) and (ii).

[0080] In some embodiments, the instructions may further comprise using the focus model to automatically position the imaging apparatus at a position of the plurality of positions. In someWSGR Docket No. 64100-742.601 embodiments, the portion of the surface may comprise one or more surfaces of a substrate 512. In some embodiments, the substrate may be coupled to a first body 518 and a second body 520 to form a well 510 between the firstbody, the secondbody and the substrate, as shown in the example of FIG. 5A. The substrate may comprise or be made of optically transparent or partially optically transparent material. In some embodiments, system instructions may be performed in a time of about 1 millisecond (ms) to about 20 ms. In some embodiments, system instructions may be performed in a time of about 1 ms to about 2 ms, about 1 ms to about 3 ms, about 1 ms to about 4 ms, about 1 ms to about 5 ms, about 1 ms to about 6 ms, about 1 ms to about 7 ms, about 1 ms to about 8 ms, about 1 ms to about 9 ms, about 1 ms to about 10 ms, about 1 ms to about 15 ms, about 1 ms to about 20 ms, about 2 ms to about 3 ms, about 2 ms to about 4 ms, about 2 ms to about 5 ms, about 2 ms to about 6 ms, about 2 ms to about 7 ms, about 2 ms to about 8 ms, about 2 ms to about 9 ms, about 2 ms to about 10 ms, about 2 ms to about 15 ms, about 2 ms to about 20 ms, about 3 ms to about 4 ms, about 3 ms to about 5 ms, about 3 ms to about 6 ms, about 3 ms to about 7 ms, about 3 ms to about 8 ms, about 3 ms to about 9 ms, about 3 ms to about 10 ms, about 3 ms to about 15 ms, about 3 ms to about 20 ms, about 4 ms to about 5 ms, about 4 ms to about 6 ms, about 4 ms to about 7 ms, about 4 ms to about 8 ms, about 4 ms to about 9 ms, about 4 ms to about 10 ms, about 4 ms to about 15 ms, about 4 ms to about 20 ms, about 5 ms to about6 ms, about 5 ms to about 7 ms, about 5 ms to about 8 ms, about 5 ms to about 9 ms, about 5 ms to about 10 ms, about 5 ms to about 15 ms, about 5 ms to about 20 ms, about 6 ms to ab out 7 ms, about 6 ms to about 8 ms, about 6 ms to about 9 ms, about 6 ms to about 10 ms, about 6 ms to about 15 ms, about 6 ms to about 20 ms, about 7 ms to about 8 ms, about 7 ms to about 9 ms, about 7 ms to about 10 ms, about 7 ms to about 15 ms, about 7 ms to about 20 ms, about 8 ms to about 9 ms, about 8 ms to about 10 ms, about 8 ms to about 15 ms, about 8 ms to about 20 ms, about 9 ms to about 10 ms, about 9 ms to about 15 ms, about 9 ms to about 20 ms, about 10 ms to about 15 ms, about 10 ms to about 20 ms, or about 15 ms to about 20 ms. In some embodiments, system instructions may be performed in a time of about 1 ms, about 2 ms, about 3 ms, about 4 ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 15 ms, or about 20 ms. In some embodiments, system instructions may be performed in a time of at least about 1 ms, about 2 ms, about 3 ms, about 4 ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, or about 15 ms. In some embodiments, system instructions may be performed in a time of at most about 2 ms, about 3 ms, about 4 ms, about 5 ms, about 6 ms, about7 ms, about 8 ms, about 9 ms, about 10 ms, about 15 ms, or about 20 ms. In some embodiments, the light beam may comprise electromagnetic wavelengths in (i) the near infra-red spectrum, (ii) the ultraviolet spectrum, (iii) the visible spectrum, or any combination thereof.WSGR Docket No. 64100-742.601

[0081] In some embodiments, the surface comprises a first surface 502 (SI) of a multi-well or microtiter plate, for example the bottom of a multi -welled plate. In some embodiments, the second surface 504 (S2) comprises a bottom surface of a well of a multi-well plate. In some embodiments, the third surface 508 (S3) comprises a fluid / air interface, wherein thefluid is suspended above the portion of the second surface 504 of the multi-well or microtiter plate.

[0082] In some cases, the first surface 502 may bebelowthe second surface 504 by about 0.8 mm to about 1 .1 mm. In some cases, the first surface 502 may be below the second surface 504 by about 0.8 mm to about 0.85 mm, about 0.8 mm to about 0.9 mm, about 0.8 mm to about 1 mm, about 0.8 mm to about 1.05 mm, about 0.8 mm to about 1.1 mm, about 0.85 mm to about 0.9 mm, about 0.85 mm to about 1 mm, about 0.85 mm to about 1 .05 mm, about 0.85 mm to about 1.1 mm, about 0.9 mm to about 1 mm, about 0.9 mm to about 1 .05 mm, about 0.9 mm to about 1.1 mm, about 1 mm to about 1 .05 mm, about 1 mm to about 1. 1 mm, or about 1.05 mm to about 1.1 mm. In some cases, the first surface 502 may bebelow the second surface 504 by about 0.8 mm, about 0.85 mm, about 0.9 mm, about 1 mm, about 1 .05 mm, or about 1.1 mm. In some cases, the first surface 502 may be below the second surface 504 by at least about 0.8 mm, about 0.85 mm, about 0.9 mm, about 1 mm, or about 1.05 mm. In some cases, the first surface 502 may be below the second surface 504 by at most about 0.85 mm, about 0.9 mm, about 1 mm, about 1.05 mm, or about 1.1 mm.

[0083] In some cases, the third surface 508 may be above the second surface 504 by about 0.2 mm to about 3.5 mm. In some cases, the third surface 508 may be above the second surface 504 by about 0.2 mm to about 0.4 mm, about 0.2 mm to about 0.8 mm, about 0.2 mm to about 1 mm, about 0.2 mm to about 1 .2 mm, about 0.2 mm to about 1 .4 mm, about 0.2 mm to about 1.8 mm, about 0.2 mm to about 2 mm, about 0.2 mm to about 2.2 mm, about 0.2 mm to about 2.8 mm, about 0.2 mm to about 3 mm, about 0.2 mm to about 3.5 mm, about 0.4 mm to about 0.8 mm, about 0.4 mm to about 1 mm, about 0.4 mm to about 1.2 mm, about 0.4 mm to about 1.4 mm, about 0.4 mm to about 1.8 mm, about 0.4 mm to about2 mm, about 0.4 mm to about 2.2 mm, about 0.4 mm to about 2.8 mm, about 0.4 mm to about 3 mm, about 0.4 mm to about 3.5 mm, about 0.8 mm to about 1 mm, about 0.8 mm to about 1.2 mm, about 0.8 mm to about 1.4 mm, about 0.8 mm to about 1.8 mm, about 0.8 mm to about2 mm, about 0.8 mm to about 2.2 mm, about 0.8 mm to about 2.8 mm, about 0.8 mm to about 3 mm, about 0.8 mm to about 3.5 mm, about 1 mm to about 1 .2 mm, about 1 mm to about 1.4 mm, about 1 mm to about 1.8 mm, about 1 mm to about 2 mm, about 1 mm to about 2.2 mm, about 1 mm to about 2.8 mm, about 1 mm to about 3 mm, about 1 mm to about 3.5 mm, about 1 .2 mm to about 1.4 mm, about 1 .2 mm to about 1.8 mm, about 1.2 mm to about 2 mm, about 1.2 mm to about 2.2 mm, about 1 .2 mm to about 2.8 mm, about 1.2 mm to about 3 mm, about 1.2 mm to about 3.5 mm, about 1.4 mm to about 1.8WSGR Docket No. 64100-742.601 mm, about 1.4 mm to about 2 mm, about 1.4 mm to about 2.2 mm, about 1.4 mm to about 2.8 mm, about 1 .4 mm to about 3 mm, about 1 .4 mm to about 3.5 mm, about 1.8 mm to about 2 mm, about 1.8 mm to about 2.2 mm, about 1.8 mm to about2.8 mm, about 1.8 mm to ab out 3 mm, about 1.8 mm to about 3.5 mm, about 2 mm to about 2.2 mm, about 2 mm to about 2.8 mm, about 2 mm to about 3 mm, about 2 mm to about 3.5 mm, about 2.2 mm to about 2.8 mm, about 2.2 mm to about 3 mm, about2.2 mm to about 3.5 mm, about2.8 mm to about 3 mm, about2.8 mm to about 3.5 mm, or about 3 mm to about 3.5 mm. In some cases, the third surface 508 may be above the second surface 504 by about 0.2 mm, about 0.4 mm, about 0.8 mm, about 1 mm, about 1 .2 mm, about 1.4 mm, about 1.8 mm, about2 mm, about2.2 mm, about 2.8 mm, about 3 mm, or about 3.5 mm. In some cases, the third surface 508 may be above the second surface 504 by at least about 0.2 mm, about 0.4 mm, about 0.8 mm, about 1 mm, about 1.2 mm, about 1.4 mm, about 1.8 mm, about 2 mm, about 2.2 mm, about 2.8 mm, or about 3 mm. In some cases, the third surface 508 may be above the second surface 504 by at most about 0.4 mm, about 0.8 mm, about 1 mm, about 1.2 mm, about 1.4 mm, about 1.8 mm, about 2 mm, about 2.2 mm, about2.8 mm, about 3 mm, about 3.5 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm.

[0084] In some embodiments, the second surface 504, the first surface 502, and the third surface 508 may be disposed or provided at a fixed position relative to each other. In some embodiments, the second surface 504, the first surface 502, and the third surface 508 may be variable in position relative to each other. In some embodiments, the difference between a position of the second surface 504 and the third surface 508 indicates a level of a fluid 506 suspended in a well of a multi-well plate. In some embodiments, the second surface 504 and the first surface 502 may be disposed or provided at a fixed position relative to a home position of the imaging apparatus 200.

[0085] In some embodiments, fitting the linear portion of the curve may comprise: (a) using a function to determine a size metric for each image corresponding to each centroid; (b) selecting the size metric which maximizes or minimizes the function; and (c) selecting a subset of the centroids corresponding to (i) the selected size metric and (ii) the size metrics between the first position and the second position of the plurality of positions, where each size metric is associated with a spot size of each image, and where the function comprises an exponential function of the spot sizes. In some embodiments, optimizing the focus metric may comprise: (a) using a function to determine a focus metric for each image of the linear portion of the curve; and (b) selecting the focus metric which maximizes or minimizes the function, where each focus metric may be associated with a spot size of each image, and where the function comprises (i) a full width half max (FWHM) function, (ii) a Brenner gradient function, or (iii) a Shannon Laplacian (SLaP) function.WSGR Docket No. 64100-742.601

[0086] In some embodiments, the instructions may comprise determining (i) a local tilt, (ii) a global tilt, or (iii) a combination of (i) and (ii), of the surface from a plane of the imaging apparatus. In some embodiments, determining the local tilt may comprise: (a) obtaining a centroid for each of the plurality of positions of the portion of the surface; (b) fitting a planar surface to the plurality of positions; and (c) determining a slope of the planar surface thereby determining the local tilt. In some embodiments, determiningglobal tilt may comprise: (a) imaging each well; (b) determine which is the optimal Cx position from focus model generation; (c) recording the z value of the apparatus as the optimal Cx determined position; (d) fitting the z values for the parametric surface model ; and (e) determining a slope of the parametric surface model at every position where a surface is expected to be imaged, thereby determining the global tilt. In some embodiments, the system instructions may comprise updating the focus model based at least on the determined local tilt or global tilt of the surface.

[0087] In some embodiments, the system instructions may comprise using a z-stage to position the imaging apparatus at the plurality of positions from the imaging apparatus. In some embodiments, the Z-stage may be configured with a travel range extending across all of the plurality of positions described herein.

[0088] In some cases, the z-stage may comprise a travel range of about 1 mm to about 15 mm. In some cases, the z-stage may comprise a travel range of about 1 mm to about 2 mm, about 1 mm to about 3 mm, about 1 mm to about 4 mm, about 1 mm to about 5 mm, about 1 mm to about 6 mm, about 1 mm to about 7 mm, about 1 mm to about 8 mm, about 1 mm to about 9 mm, about 1 mm to about 10 mm, about 1 mm to about 12 mm, about 1 mm to about 15 mm, about 2 mm to about 3 mm, about 2 mm to about 4 mm, about 2 mm to about 5 mm, ab out 2 mm to about 6 mm, about 2 mm to about 7 mm, about 2 mm to about 8 mm, about 2 mm to about 9 mm, about 2 mm to about 10 mm, about 2 mm to about 12 mm, about 2 mm to about 15 mm, about 3 mm to about 4 mm, about 3 mm to about 5 mm, about 3 mm to about 6 mm, about 3 mm to about 7 mm, about 3 mm to about 8 mm, about 3 mm to about 9 mm, about 3 mm to about 10 mm, about 3 mm to about 12 mm, about 3 mm to about 15 mm, about 4 mm to about 5 mm, about 4 mm to about 6 mm, about 4 mm to about 7 mm, about 4 mm to about 8 mm, about 4 mm to about 9 mm, about 4 mm to about 10 mm, about 4 mm to about 12 mm, about 4 mm to about 15 mm, about 5 mm to about 6 mm, about 5 mm to about 7 mm, about 5 mm to about 8 mm, about 5 mm to about 9 mm, about 5 mm to about 10 mm, about 5 mm to about 12 mm, about 5 mm to about 15 mm, about 6 mm to about 7 mm, about 6 mm to about 8 mm, about 6 mm to about 9 mm, about 6 mm to about 10 mm, about 6 mm to about 12 mm, about 6 mm to about 15 mm, about 7 mm to about 8 mm, about 7 mm to about 9 mm, about 7 mm to about 10 mm, about 7 mm to about 12 mm, about 7 mm to about 15 mm, about 8 mm to about 9 mm, about 8 mm to about 10 mm, about 8 mm toWSGR Docket No. 64100-742.601 about 12 mm, about 8 mm to about 15 mm, about 9 mm to about 10 mm, about 9 mm to about 12 mm, about 9 mm to about 15 mm, about 10 mm to about 12 mm, about 10 mm to about 15 mm, or about 12 mm to about 15 mm. In some cases, the z-stage may comprise a travel range of about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 12 mm, or about 15 mm . In some cases, the z -stage may comprise a travel range of at least about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about ? mm, about 8 mm, about 9 mm, about 10 mm, or about 12 mm. In some cases, the z-stage may comprise a travel range of at most about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about ? mm, about 8 mm, about 9 mm, about 10 mm, about 12 mm, or about 15 mm.

[0089] In some embodiments, the z-stage may be configured with a depth of focus of the plurality of positions from the image apparatus comprising a range of up to about 5 pm with an accuracy of about 10% of the depth of focus.

[0090] In some cases, the local tilt of a surface may be obtained from a plurality of surface focal ranges or surface focus models generated for one or more positions of the surface, as shown in FIGs. 9B-9C. In some cases, at least three positions or regions of a surface may be analyzed to determine a position of the surface or a focal position of the imaging apparatus with respect to the surface to focus the imaging apparatus on the surface. In some cases, the optimized focal position of the imaging apparatus may be determined from analyzing a linear relationship between the position of the surface or focal position of the one or more portions of the surface as shown in the example of FIG. 9B. In some cases, local tilt may be determined by measuring a slope of a plane fitted to the at least three positions or regions of a surface, e.g., a surface of a well, as shown in the example of FIG. 9C . In some cases, the z axis shown in the example of FIG. 9C may comprise the z-travel or distance between the imaging apparatus and the surface imaged where the imaging apparatus is focused on the surface imaged, described herein.

[0091] In some embodiments, the system instructions may comprise using the calibrated image apparatus to autofocus one or more particles positioned proximate to or immobilized to the portion of the surface. In some embodiments, the one or more particles may comprise amplification products coupled to fluorescently labeled polynucleotides. In some embodiments, the amplification products and the fluorescently labeled polynucleotides coupled thereto may be suspended in a buffer. In some embodiments, the coupling may comprise hybridization of the amplification products to the fluorescently labeled polynucleotides. In some embodiments, the coupling may comprise covalent bonds. In some embodiments, the coupling may comprise non- covalent bonds. In some embodiments, the non-covalent bonds may include molecular interactions such as van der Waals forces, ionic forces and hydrogen bonding.WSGR Docket No. 64100-742.601

[0092] In some embodiments, the second surface 504 may comprise an array of one or more other surface portionsof the surface. In some embodiments, the surface may compriseone or more other surface portions of a microtiter or multi-well plate. In some embodiments, the surface may comprise one or more other surface portions of a 96-well plate, a 384-well plate, or a 1536-well plate. In some embodiments, the portion of the surface may be patterned as a shape comprising (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a geometric shape, or (v) any combination of (i) - (iv). In some embodiments, the portion of the surface may comprise a surface area of about 0.02 cm2, about 0.03 cm2, about 0.05 cm2, about 0.08 cm2, about 0.1 cm2, about 0.2 cm2, about 0.3 cm2, about 0.4 cm2, about 0.5 cm2, about 0.6 cm2, about 0.7 cm2, about 0.8 cm2, about 0.9 cm2, about 1.0 cm2, or greater.Methods for Calibrating an Imaging Apparatus

[0093] Aspects of the disclosure describe a method of determining a focal correction of an imaging apparatus 400, as illustrated in the example of FIG. 4. In some instances, the method 400 may comprise activating a light source 401; directing an emittedbeam of the light source through an objective lens to one or more surfaces 402; detecting reflected light from the one or more surfaces directed back through the objective lens to an autofocus module 404; acquiring an image of the detected reflected light 406; and analyzing the image and determining a focal correction of the imaging apparatus 408. In some cases, the light source may comprise a laser. In some instances, the light source may comprise one or more light emitting diodes. In some cases, the light source may emit light with one or more center wavelengths of light. In some instances, the light source may deliver and / or provide light of a range of wavelengths, for example light that encompasses the visible spectrum of light. In some cases, the image, as shown in the example of FIG. 5B may comprise a detected reflected light source from the one or more surfaces or interfaces simultaneously as the imaging apparatus is translated towards the one or more surfaces or interfaces.

[0094] In some cases, a detector of the imaging apparatus may detect a reflected light source from the one or more surfaces (SI, S2 and S3) in a detectable range (528, 530, 532) of z-positions of a z-stage coupled to the imaging apparatus. In some instances, the methods and / or systems described herein may determine a linear range of focuspositions (522, 524, 526) on a surface from two or more images of the reflected beam from the surface, as shown in the example of FIG. 5B. In some embodiments, the detectable range (528, 530, 532) may comprise a first surface 502 detectable range 528, the second surface 504 detectable range 530, the third surface 508 detectable range 532, or a combination of one or more thereof detectable range(s) . In some cases, the linear range of focus positions as illustrated in the example of FIG.26 (2622, 2624, 2626) may comprise the first surface linear range of focus positions 2622, the second surface linear range of focusWSGR Docket No. 64100-742.601 positions 2624, the third surface linear range of focus positions 2626, or a combination of one or more thereof. In some instances, one or more upper and lower ranges of error (2634, 2636, 2638) may be determined for the one or more linear ranges of focus positions (2622, 2624, 2626, respectively) of the one or more surfaces. In some cases, the upper and lower ranges of error (2634, 2636, 2638) may comprise an error range of up to about 5% greater than or less than the identified linear range of focus positions (2622, 2624, 2626) of the one or more surfaces.

[0095] In some cases, the light that is delivered through the objective lens may be reflected by the surface as defined by the z focal length of the objective lens and directed back through the objective lens to the autofocus module. In some embodiments, the z-stage movement of the movable objective lens can be extended from zero to about a 1000 pm in order to focus the objective lens on the surface. For example, in some cases, the z-stage may be moved such that the focal length of the objective lens is directed to the bottom of a substrate (SI). In some cases, the z-stage may be moved such that the focal length of the imaging apparatus is directed to the interface between the top of a substrate and an aqueous solution (S2). In some instances, the z- stage may be moved such that the focal length of the imaging apparatus may be directed to the interface between the aqueous solution and the air (S3). In some instances, the z-stage movement range may be from about 1 ,000 pm to about 5,000 pm. In some cases, the z -stage movementrange may be from about 1,000 pm to about 200 pm. In some cases, the z-stage movement can be measured from a user-defined origin of coordinate system.

[0096] Aspects of the disclosure describe a method of calibrating an imaging apparatus that may be implemented as one or more algorithms or one or more processes implemented as software on one or more processors of the systems described herein. In some embodiments, the method of calibrating the imaging apparatus may comprise methods or software modules of instrument calibration 1100, methods of tip and tilt 1200, or methods of functional verification (1300, 1400, 1500), as illustrated in the example of FIG. 10.

[0097] In some embodiments, the instrument calibration method 1100, as shown in the example of FIG. 11, may determine a tip or tilt of a target surface based on a slope of a plane generated from a plurality of measurements of one or more position(s) of one or more portions of the target surface, e.g., a surface with one or more pin hole structural features. The instrument calibration may comprise homing the tip-tilt motors, described herein, and loading the target surface plate 1111. The instrument calibration may further comprise measuring at least about 4 portions of the surface, e.g., 4 comers of the surface with the imaging apparatus 1112. The instrument calibration may further comprise fitting a planar tilt of the loaded surface based at least on the 4 comers of the target surface measured 1113. The instrument calibration may further comprise generating or determining planar tilt of the plane such that the plane comprises a zero slope 1114. In someWSGR Docket No. 64100-742.601 embodiments, the instrument calibration method 1100 may comprise confirmatory global tilt of the target surface by obtaining or acquiring one or more sets of images of a reflected beam at a plurality of positions of the imaging apparatus with respect to the target surface or at one or more regions across the target surface with the imaging apparatus to determine imaging tilt 1115. The instrument calibration method 1100 may further comprise interpolating the best tilt of the plane of the target surface across the plurality of positions or regions across the target surface 1116. The instrument calibration method 1100 may further comprise setting or actuating the tip-tilt motors, described herein, to adjust the slope tip or tilt of the target surface based at least on the 4 comer planar tilt measurements 1117. The instrument calibration method 1100 may further comprise obtaining or acquiring one or more sets of images of the reflected beam atthe plurality of positions of the imaging apparatus with respectto the target surface 1118. In some embodiments, the method 1110 may comprise analyzing the one or more sets of images of the reflected beam off of the target surface to determine if less than about 1 milliradian (mrad) of tip or tilt of the target surface has been determined 1119. In some embodiments, if less than about 1 mrad of tip or tilt is determined, then the tip or tilt values of the tip-tilt motors, e.g., the theta X motor rotation or the theta Y motor rotation, described herein, may be saved 1120. In some embodiments, if less than about 1 mrad tip or tilt of the surface is not determined from the analyzed one or more sets of images of the reflected beam off of the target surface, then the calibration routine may be determined to have failed 1121. In the circumstance where the calibration routine has failed, the method may begin at the global tip or tilt determination (1111-1114).

[0098] Aspects of the disclosure provide a method for calibrating the imaging apparatus for autofocus 2200, as illustrated in the example of FIG. 22. In some embodiments, the method 2200 may comprise: directing a light beam from a light source to a portion of a surface positioned proximate to a plurality of positions from a first imaging apparatus 2202, receiving a reflected portion of the light beam onto an imaging detector of the first imaging apparatus to form an image at each position of the plurality of positions 2204, fitting a linear portion of a relationship (e.g., curve) between the image from the first imaging apparatus and the objection translator position for a subset of the plurality of positions 2206, and optimizing a focus metric obtained from the linear portion of the relationship 2208. Calibrating the instrument apparatus further comprises accessing a second imaging apparatus 2210 and repeating steps 2202-2208 for the second imaging apparatus. A method for calibrating the imaging apparatus for autofocus further comprises generating a focus model based at least on the optimized focus metric from the first and second imaging systems thereby calibrating the second imaging apparatus 2212-2214 for, for example, imaging the detection events for fluorescently labeled products. In some embodiments, the method may comprise using the focus model to automatically position the imaging apparatus at a positionWSGR Docket No. 64100-742.601 of the plurality of positions. In some embodiments, the method may comprise updating the focus model by repeating (a) - (d) for each of one or more other portions of the surface. In some embodiments, the method may be performedin a time, where the time may comprise atmost about 500 ms, at most about400 ms, at most about 300 ms, at most about 200 ms, at most about 100 ms, or less. In some embodiments, the time to perform the method 2200 may be about 200 ms. In some embodiments, the light beam may comprise EM wavelengths in (i) the NIR spectrum, (ii) the UV spectrum, (iii) the visible spectrum, or any combination thereof.

[0099] In some embodiments, a distance between the first position and the second position of the plurality of positions may comprise a distance of at most about 100 pm, at most about 90 pm, at most about 80 pm, at most about 70 pm, at most about 60 pm, at most ab out 50 pm, at most about 40 pm, at most about 30 pm, at most about 20 pm, or at most about 10 pm. In some embodiments, the distance may be about 40 pm. In some embodiments, the method 2200 may comprise (i) moving the imaging apparatus continuously at a rate between each position of the plurality of positions, (ii) moving the imaging apparatus periodically at a period between each position of the plurality of positions, or (iii) a combination of (i) and (ii).

[0100] In some embodiments, the surface may comprise a bottom surface of a well of a microtiter or multi-well plate. In some embodiments, the first surface may comprise a bottom surface of a microtiter plate or multi-well plate. In some embodiments, the third surface may comprise a top surface of a fluid suspended above the portion of the surface. In some embodiments, the first surface may be up to about 10 mm below the surface. In some embodiments, the third surface may be up to about 10 mm above the surface. In some embodiments, the first surface and the third surface may be disposed or provided at a fixed position relative to each other. In some embodiments, the surface, the first surface, or the third surface may be disposed or provided at a variable position relative to each other. In some embodiments, a difference in a position between the surface and the third surface may indicate a level of the fluid suspended above the portion of the surface. In some embodiments, the surface, the first surface, or the third surface may be provided or disposed at a fixed position relative to a home position of the imaging apparatus.

[0101] In some embodiments, the surface may comprise an array of one or more other surface portions of the surface. In some embodiments, the surfacemay comprise one or more other surface portions of a microtiter plate or multi-well plate. In some embodiments, the surface may comprise one or more other surface portions of a 96-well microtiter plate, a 384-well microtiter plate, or a 1536-well microtiter plate. In some embodiments, a portion of the surface is patterned as a shape comprising (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a geometric shape, or (v) any combination of (i) - (iv). In some embodiments, the portion of the surface may comprise a surface area of about 0.02 cm2, about 0.03 cm2, about 0.05 cm2, about 0.08 cm2, about 0.1 cm2,WSGR Docket No. 64100-742.601 about 0.2 cm2, about 0.3 cm2, about 0.4 cm2, about 0.5 cm2, about 0.6 cm2, about 0.7 cm2, about 0.8 cm2, about 0.9 cm2, about 1.0 cm2, or greater.

[0102] In some embodiments, the method may comprise: (a) performing (a) - (d) for a portion of a first surface, wherein the first surface is below the surface; (b) performing (a) - (d) for a portion of a third surface, wherein the third surface is above the surface; and (c) updating the focus model based at least on (a) and (b). In some embodiments, fitting the linear portion of the curve may comprise: (a) using a function to determine a size metric for each image corresponding to each centroid; (b) selecting the size metric which maximizes or minimizes the function; and (c) selecting a subset of the centroids corresponding to (i) the selected size metric and (ii) the size metrics between the first position and the second position of the plurality of positions, where each size metric may be associated with a spot size of each image, and where the function may comprise an exponential function of the spot sizes. In some embodiments, optimizing the focus metric may comprise: (a) using a function to determine a focus metric for each image corresponding to each centroid of the linear portion of the curve; and (b) selecting the focus metric which may maximize or minimize the function, where each focus metric is associated with a spot size of each image, and where the function may comprise (i) a FWHM function, (ii) a Brenner gradient function, or (iii) a SLaP function.

[0103] In some embodiments, the method 2200 may comprise determining (i) a local tilt, (ii) a global tilt, or (iii) a combination of (i) and (ii), of the surface from a plane of the imaging apparatus. In some embodiments, determining the local tilt may comprise: (a) obtaining a centroid for each of the plurality of positions of the portion of the surface; (b) fitting a planar surface to the plurality of positions; and (c) determining a slope of the planar surface thereby determining the local tilt. In some embodiments, determining the global tilt may comprise: (a) obtaining a centroid for each of the plurality of positions at a plurality of portions across the surface; (b) fitting a quadratic surface to the plurality of positions at the plurality of portions of the surface; and (c) determining a slope of the quadratic surface thereby determining the global tilt. In some embodiments, the method 2200 may comprise updating the focus model based at least on the determined local tilt or global tilt of the surface.

[0104] In some embodiments, the method 2200 may comprise using a z-stage to position the imaging apparatus at the plurality of positions from the imaging apparatus. In some embodiments, the z-stage may be configured with a travel range of the plurality of positions from the image apparatus comprising a range of up to about 5 mm. In some embodiments, the z-stage may be configured with a capture range of the plurality of positions from the image apparatus comprising a range of up to about 2 mm. In some embodiments, the z-stage may be configured with a depthWSGR Docket No. 64100-742.601 of focus of the plurality of positions from the image apparatus comprising a range of up to about 5 pm with an accuracy of about 10% of the depth of focus.

[0105] In some embodiments, the method 2200 may comprise using the calibrated image apparatus to autofocus on one or more particles positioned proximate to or immobilized to the portion of the surface. In some embodiments, the one or more particles may comprise amplification products coupled to fluorescently labeled polynucleotides. In some embodiments, the amplification products or the fluorescently labeled polynucleotides coupled thereto may be suspended in a buffer. In some embodiments, the coupling between the amplification products and the fluorescently labeled polynucleotides may comprise hybridization of the amplification products to the fluorescently labeled polynucleotides. In some embodiments, the coupling may comprise covalent bonds. In some embodiments, the coupling may comprise non-covalent bonds, for example van der Waals forces, ionic forces, or hydrogen bonding.

[0106] Aspects of the disclosure describe a method of fitting a measured position of a surface, e.g. , one or more wells of a multi-well plate or a microtiter plate, to a quadratic model to determine global tip-tilt of one or more surfaces or interfaces, as described herein, and as shown in the examples of FIGs. 12A-12B. In some cases, the methods of tip and tilt 1200 may comprisehoming thetip-tilt motorsto a calibrated home position 1205,as describedherein and measuring a position of a plurality of regions across the surface, e.g., a surface of one or more wells of a multi -well plate or a microtiter plate with an imaging apparatus 1206. In some cases, the plurality of regions comprise about 10 regions to about 26 regions. In some cases, the plurality of regions comprise about 10 regions to about 12 regions, about 10 regions to about 14 regions, about 10 regions to about 16 regions, about 10 regions to about 18 regions, about 10 regions to about20 regions, about 10 regions to about 22 regions, about 10 regions to about 24 regions, about 10 regions to about 26 regions, about 12 regions to about 14 regions, about 12 regions to about 16 regions, about 12 regions to about 18 regions, about 12 regions to about 20 regions, about 12 regions to about 22 regions, about 12 regions to about 24 regions, about 12 regions to about 26 regions, about 14 regions to about 16 regions, about 14 regions to about 18 regions, about 14 regions to about 20 regions, about 14 regions to about 22 regions, about 14 regions to about 24 regions, about 14 regions to about 26 regions, about 16 regions to about 18 regions, about 16 regions to about 20 regions, about 16 regions to about 22 regions, about 16 regions to about 24 regions, about 16 regions to about 26 regions, about 18 regions to about 20 regions, about 18 regions to about 22 regions, about 18 regions to about 24 regions, about 18 regions to about 26 regions, about 20 regions to about 22 regions, about 20 regions to about 24 regions, about 20 regions to about 26 regions, about 22 regions to about 24 regions, about 22 regions to about 26 regions, or about 24 regions to about 26 regions. In some cases, the plurality of regions comprise about 10 regions,WSGR Docket No. 64100-742.601 about 12 regions, about 14 regions, about 16 regions, about 18 regions, about 20 regions, about 22 regions, about 24 regions, or about 26 regions. In some cases, the plurality of regions comprise at least about 10 regions, about 12 regions, about 14 regions, about 16 regions, about 18 regions, about 20 regions, about 22 regions, or about 24 regions. In some cases, the plurality of regions comprise at most about 12 regions, about 14 regions, about 16 regions, about 18 regions, about 20 regions, about 22 regions, about 24 regions, or about 26 regions.

[0107] In some instances, the measured position of the at least 24 regions across the surface may be validated 1207, where the validation comprises determining if there are at least 20 measurements of the position of the surfaces across the at least 24 regions measured that did not lead to a failure, as described herein. In some cases, if at least 20 measurements did not succeed, then a failure 1208 may be determined by the methods oftip andtilt 1200. In some embodiments, if at least 20 measurements have succeeded then the at least 20 measurements of the position of the surface may be fitted to a quadratic model 1209, as shown in the example of FIG. 12B. In some embodiments, the quadratic model may be analyzed for fit by analyzing the R2value of the fit to determine whether or not the R2value of the fit is at least about 0.95 1210. In some embodiments, if the value of the R2surface fit is found to be at least about 0.95, then the model curvature for the fitted model may be set as the tilt model for the surface 1211. In some embodiments, if the value of the R2of the surface fit is found not to be at least about 0.95 then a failure 1208 may be noted or recorded.

[0108] Aspects of the disclosure describe a functional verification method 1300 of validating the movement of tip-tilt motor(s) in a system, as described herein, as shown in the examples of FIGs. 13A-13B. In some cases, the functional verification method 1300 may comprise loading a well plate or an optical target into a system 1301, as described herein, initializing the tip-tilt motors to a home position 1306. In some instances, following this initialization, the tip-tilt motors may be moved across their full range of motion while a corresponding encoder value(s) are recorded 1307. In some instances, the recorded motion of the motor(s) movement may then be plotted against the encoder values 1308, as shown in the example of FIG. 13B. In some embodiments, upon analyzing the motor movements against the encoder values, the encoder values may be validated 1309

[0109] In some instances, the method(s) may include storing or analyzing the obtained encoder readings for future reference or for executing specific tasks. In some embodiments, if the encoder is the same with the expected motor positions throughout its range, the method may consider the tip-tilt motor and encoder as functioning correctly . In some embodiments, if, however, a consistent discrepancy is noted between the encoder readings and the motor positions, the system might record a failure, and the required corrective measures may be invoked.WSGR Docket No. 64100-742.601

[0110] For example, as illustrated in the example of FIG. 14, systems herein may be configured to perform a method 1400 of: loading 1410 a target, e.g., a multi-well plate, microtiter plate, or optical target; homing 1411 to ensure the tip-tilt encoder is set to the zero position; measuring 1412 a plurality of Z positions of edge wells; fitting 1413 the measurements to a plane; and determining whether all iterations have been measured 1414. In some embodiments, if all iterations have not been measured, the method may include moving 1417 the stage 100 in theta X or theta Y (e.g., in + / - 3 mrad increments) and beginning the method at 1412. In some embodiments, if all interactionshave been measured, the method may include verifying 1415 tilt changes match Z position changes of the consumable (e.g., a multi-well plate, or a microtiter plate); and verifying 1416 that the encoder values of tip-tilt match values sent to the tip-tilt.

[0111] As illustrated in the example of FIG. 15, systems herein may be configured to performa method of determining a localized tip or tilt of a surface 1500. In some cases, the method 1500 may comprise: homing 1520 the z-stage of the imaging apparatus; movingthe imaging apparatus to a first position (e.g., xl, yl) on a surface 1521; capturing an image of a reflected beam off of the surface and calculating a centroid of the reflected beam 1522; moving the z- stage of the imaging apparatus by 100 pm 1523; and repeating the capture of the reflected beam and calculating the centroid of the reflected beam 1524. In some instances, once the z-stage of the imaging apparatus has translated through the entire z travel range, the method may comprise: fitting a depth model (e.g., a linear fit) to the position of the centroid of the reflected beam across the z travel range 1525; moving to a mean range of the linear fit 1526; capturing a reflection of a light beam reflected off of the surface and calculating a centroid position of the reflected beam 1527; moving the imaging apparatus to a second position (e.g., x2, y2) on the surface 1528; capturing a reflection of a lightbeam reflected off of the surface and calculating a centroid position of the reflected beam 1529; movingthe imaging apparatus to a third position (e.g., x3, y3) on the surface 1530; processing a cross product of the normal vector and returning a tip-tilt 1531; actuating a tip-tilt motor to de-tilt the surface 1532; and verifying the de-tilt 1533.

[0112] As illustrated in the example of FIG. 16, systems herein may be configured to perform an autofocus method. In some cases, the autofocus method may comprise conducting a surface range search and computing or processing a linear range of an identified surface 1700, identifying a focus depth model 1800, generating the focus model 1900, initiating or conducting an autofocus test of autofocusing the imaging apparatus on the surface 2000, and autofocusing the imaging apparatus on the surface 2100.

[0113] As illustrated in the example of FIG. 17, systems herein may be configured to performa method 1700 of determining a start and stop position of a focus model for a surface, described herein. In some cases, the method 1700 may comprise receiving or providing input parameters,WSGR Docket No. 64100-742.601 e.g., z travel range of a z-stage coupled to the imaging apparatus, coarse stepping distance of the z-stage, fine stepping distance of the z-stage, the imaging apparatus camera parameters, a debug mode flag, or any combination thereof 1710, loading and / providing a multi-well or a microtiter plate into the imaging or fluidic system and starting data acquisition to determine the focus model forthe surface ofthe multi-well or microtiter plate 1711, andhomingthe z-stage, loweringthe z- stage to the lowest position, and enabling an output of a beam of light from a light source (e.g., a 785 nm laser) 1712. Continuing with FIG. 17, method 1700 further comprises capturing or detecting an image of a first reflection of the beam of light reflected from the surface 1713, calculating or processinga centroid position for the reflected beam 1714; detecting or determining a second reflection of the beam of light reflected from a second surface 1715, and translating a unit of course stepping distance of the z-stage 1716 and beginning the method again at capturing an image of the first reflection of the beam of light from the surface 1713. In some cases, the laser may be turned off 1717 if the imaging system identifies 1715 within a z travel range of greater than about 200 pm where (i) a first image of the reflected beam of light shows or indicates the first reflection of the beam of lightreflectedfrom the surface andthe second reflection ofthe beam of light reflected from the second surface, and (ii) a second image, acquired subsequent to the first image, that shows or indicates the first reflection of the beam of light reflected from the surface. The method 1700 may comprise determining a start position of the surface from a z-stage position where the first reflection of the beam of light from the surface and the second reflection of the beam light from the second surface are detected 1718. In some cases, the method 1700 may determine a stop position of the surface from a z-stage position where the reflection of the beam of light from the surface is detected 1718. In some cases, the method 1700 may output one or more parameters 1719 of the approximate start ofthe position ofthe surface within the focus model, the approximate stop position of the surface within the focus model, a change in a state machine as described in 1715. In some cases, if a debug mode is activated, a table indicating the z-stage z position and centroid positions for the surface and the second surface, or any combination thereof, may be generated and outputted.

[0114] As illustrated in the example of FIG. 18, systems herein may be configured to perform a method 1800 to determine a focus model of a surface from a plurality of centroid positions of a beam reflected off of the surface. In some cases, the method 1800 may comprise receiving or providing input parameters, e.g., the z position of the z-stage indicative of the start position of the surface (e.g., S2_Z_start), the z position of the z-stage indicative of the stop position (e.g., S2_Z_stop) of the surface; the z-stage fine step size, the imaging apparatus camera parameters, a debug mode flag, or any combination thereof 1801. Continuing with FIG. 18, the method 1800 further comprises calculating the number of steps (e.g., rounding (S2_Z_stop - S2_Z_Start) / WSGR Docket No. 64100-742.601(0.5*the z-stage fine step size)) 1802, moving the z-stage to the z position indicative of the start position of the surface 1803, enabling or turning on a laser (e.g., 785 nm laser) 1804, capturing an image of the reflection of abeam ofthe laserfromthe surface 1805, and updatingthe state machine 1806, described herein. Continuing with FIG. 18, method 1800 further comprises calculating a left centroid position of the reflected beam ofthe laser from the surface 1807, calculating a beam size (e.g., beam diameter) of the centroid of beam ofthe light source reflected by the surface 1808, moving a step of the fine step size ofthe z-stage 1809, disabling or turning off the laser 1810, and assembling the z position corresponding to (i) the detected centroid of the reflected beam of the light source reflected off of the surface and (ii) the centroid spot size 1811. Method 1800 of BIG. 18 further comprises fitting a parabolic model to the centroid spot size and returningthe z position of the z-stage corresponding to the minimum of the centroid spot size 1812, fitting a random sample consensus (RAN SAC) model over a linear range of the detected centroid of the reflected beam of the laser from the surface and return an upper surface bound of the z-position of the z- stage, lower surface bound of the z-position of the z-stage, the R2fit of the model, or any combination thereof 1813, and outputting parameters, e.g., the upper surface bound of the z- position of the z-stage, lower surface bound of the z-position of the z-stage, or the z-position of the z-stage that provides the optimal focus of the imaging apparatus on the surface 1814.

[0115] As illustrated in the example of FIG. 19, systems herein may be configured to perform a method 1900 of generatingafocusmode.In some cases, the method 1900may comprisereceiving or providing input parameters, e.g., the autofocus image offset, the z position of the z-stage indicative of the start position of the surface, the z position of the z-stage indicative of the stop position of the surface, the z position of the z-stage indicative of the optimized focus ofthe surface forthe imaging apparatus, the focus model generation range, the focu s model generation step size, the focus model generation green Boolean parameter, the focus model generation red Boolean parameter, the debugmodeflag, or any combination thereof 1901. Method 1900 of FIG. 19 further comprises calculating a z start position of the z-stage and z end position of the z-stage (e.g., z start position = ((the z position of the optimized focus of the surface for the imaging apparatus + the autofocus image offset) - the focus model generation range) / 2 and z end position = (the z po sition of the optimize focus of the surface for the imaging apparatus + the autofocus image offset + the focus model generation range) / 2 1902, calculating the number of steps (e.g., the focus model generation steps = round(focus model generation range / focus model generation step size) 1903, moving the z-stage to the z-start position 1904, enabling or turning on a first laser light source (e.g., a 785 nm laser) 1905, and capturing an image of a beam of the first laser light source reflected off of the surface 1906. Method 1900 of FIG. 19 further comprises calculating a left centroid position of the image of the beam of the first laser light source reflected off of the surfaceWSGR Docket No. 64100-742.6011907, disabling an emission of the first laser light source or turning off the first laser light source1908, determining or detecting a flag indicating to turn on a second laser light source 1909, enabling or turning on the second laser light source (e.g., a 639 nm laser) 1910, capturing or detecting an image of second laser light source reflecting off of the surf ace in channel 3 or channel 4 of a detector 1911, and calculating or determining a FWHM of the reflected beam diameter 1912. Continuing with FIG. 19, the method further comprises disabling or turning off the second laser light source 1913, determining or detecting a flag indicating to turn on a third laser light source 1914, enabling or turning on the third laser light source (e.g., a 530 nm laser) 1915, capturing or detecting an image of third laser light source reflecting off of the surface in channel 1 or channel 2 of a detector 1916, and calculating or determining a FWHM of the reflected beam diameter 1917. Continuing with FIG. 19, the method further comprises disabling or turning off the third laser light source 1918, moving the z-stage by a step size of the focus model generation step size 1919, and repeatingthe method starting at 1905 across the focus model generation range. In some cases, once the centroid position(s) of a reflected beam of the first laser, the second laser, and the third laser reflected off of the surface have been determined across the focus model range, the focus model may be assembled 1920 with the z position of the z-stage, the centroid positions of the first laser, the second laser, and the third laser detected by the FWHM in channels 1, 2, 3, and 4. In some cases, the focus model may be generated from the assembled focus model by calculating the FWHM at the optimized focus, the z-position of the z-stage at the optimized focus, the centroid position of the reflected beam of the first, second, or third laser at the optimized focus of the surface, and the fit parameters 1921. In some cases, the method 1900 may output FWHM at the optimized focus, the z-position of the z-stage at the optimized focus, and the centroid position of a reflected beam of the first, second, or third laser at the optimized focus of the surface 1922. In some instances, if the debug mode flag is equal to 1, a focus model generation fit parameter may be provided in a table for the FWHM of channel(s) 1, 2, 3, or 4 1922.

[0116] As illustrated in the example of FIG. 20, systems herein may be configured to perform a method 2000 of testing autofocus of an imaging apparatus. In some cases, the method 2000 may comprise providingorreceivingone ormore inputparameters, e.g., az position of a z-stage where the imaging apparatus is optimally focused on a surface, the centroid position of a reflected beam off of the surface providing the optimal focus, the z position of the z-stage corresponding to focus on the upper bound of the surface, the z position of the z-stage corresponding to focus on a lower bound of the surface, a test list of relative z positions, a gain plot of centroid position plotted against the z position of the z-stage, the number of autofocus cycles, the test z-step size, the number of test z steps, or any combination thereof 2001, moving the z-stage to the position of the optimized focus of the imaging apparatus focusing on the surface 2002, conducting a relativeWSGR Docket No. 64100-742.601 movement of the imaging apparatus 2003, enabling or turning on a first light source (e.g., a 785 nm laser) to emit a beam of light 2004, and capturing or detecting an image of a reflected beam of the first light source off of the surface 2005. Method 2000 further comprises calculating or processing the image of the reflected beam of the first light source to determine a left centroid position 2006, and determining if the centroid position is within a range of the centroid positions between a lower centroid z-position bound and an upper centroid z-position bound 2007. In some cases, if the centroid position is outside the range of the centroid positions between the lower centroid z-position bound and the upper centroid z-position bound, the method 2000 may return out of bound and exit the method. In some instances, if the centroid position is within the range of the centroid positions between the lower centroid z-position bound and the upper centroid z- position bound then the method 2000 may comprise determining a delta z from the gain plot and z-position of the z-stage providing optimal focus of the imaging apparatus on the surface by interpolation 2008, and conducting or issuing a relative move of the z-stage 2009. In some cases, the method operations 2005-2009 may be repeated after issuing the relative move command of the z-stage for the number of autofocus cycles. In some cases, the method 2000, after completing the autofocus cycle, may comprise capturing an image of the reflected beam of the first light source off of the surface 2010, calculating a centroid position of the reflected beam of the first light source 2011, disabling or turning off the first light source 2012, and conductingthe following method operations 2014-2021 for each cycle of the autofocus cycle, z-position of the z-stage, or centroid position of the reflected beam of the first light source 2013. In some embodiments, the method 2000 may comprise moving the z-stage by a distance (e.g., equal to rounding ((number of steps / 2) + the step sizes)) 2014, turning on or enabling a second light source (e.g., a 530 nm laser)2015, capturing an image of a beam of light of the second light source reflected off of the surface2016, moving the z-stage by a test z step size 2017, and repeating the method starting at operation 2014. In some cases, the method 2000 may comprise capturing or processing a FWHM of the beam of light of the second light source reflected off of the surface 2018, disabling or turning off the second light source 2019, calculating the FWHM of the reflected beam of the second light source at the optimal focus and the z-position of the z-stage 2020, calculating the delta z position focus (e.g., z position as a function of cycle number - the z position of the optimal focus) 2021, and continuingto anextposition in the test list 2022 and iterating operations 2002-2021 of method 2000. In some instances, the method 2000 may output 2023 one or more parameters for each focus model generation operation, each autofocus cycle iteration, or any combination thereof. In some cases, the one or more parameters may comprise cycle number, z -position, centroid position, timing, or a table, where the table may comprise, for each focus model generation operation, a z - position as a function of cycles, the z-position for optimal focus, or the delta z at the optimal focus.WSGR Docket No. 64100-742.601

[0117] As illustrated in the example of FIG. 21, systems herein may be configured to perform a method of autofocusing an imaging apparatus 2100. In some cases, the method 2100 may comprise providing or receiving one or more input parameters, e.g., a z-position of a z-stage corresponding to the optimal focus of the imaging apparatus on the surface, the centroid position of a reflected beam of a light source off of a surface at the optimal focus of the imaging apparatus, the z-position of the z-stage indicating an upper bound of focus on the surface, the z position of the z-stage indicating a lower bound of focus on the surface, the gain plot of the centroid position against the z-position of the z-stage, the number of autofocus cycles, the debug mode flag, or any combination thereof 2101, enabling or turning on the light source 2102 to emit a beam of light, capturing an image of the beam of light of the light source reflected off of the surface 2103, calculatingthe left centroid position of the reflectedbeam of light reflected off of the surface 2104, and determining or processing the position of the centroid of the reflected beam of light to determine if the centroid position is within the lower and upper bounds of the centroid positions 2105. In some embodiments, if the centroid position is outside of the lower and upper bounds of the centroid positions, then the method 2100 may return an out of bounds output and exit the method. In some embodiments, if the centroid position is within the lower and upper bounds of the centroid positions, then the method 2100 may comprise determining a delta z from the gain plot and z position of the z-stage by interpolation 2106, and issuing a calculated relative move command to move the z-position of the z-stage 2107. In some cases, the method 2100 may comprise conducting operations 2103-2107 for each autofocus cycle. In some cases, if the debug mode flag is set to a true or an on position, the method 2100 may comprise capturing or detecting an image of the reflected beam of the light source reflected off of the surface 2108, and calculating or determining a position of the centroid of the reflectedbeam of the light source reflected off of the surface 2109. The method 2100 further comprises disabling or turning off the light source 2110, and outputting one or more parameters 2111, e.g., a list of relative z-position movement of the z-stage. In some cases, if the debug mode is on or set to true (e.g., a value of 1), the method 2100 may output 2111 a table indicating the cycle number, z-position of the z-stage, and centroid position of the reflected beam of the light for each autofocus cycle.

[0118] As illustrated in the example of FIG. 25, systems herein may be configured to perform a method 2500 for determining a position of one or more surfaces from an image (e.g., one or more beam spot centroid images and / or beam spot centroid image data, described herein) of a reflected beam of a light source directed and / or provided to the one or more surfaces. In some cases, the method 2500 may comprise the operations of: opening and / or receiving centroid image data of the one or more images of the beam of the light source reflected from the one or more surfaces 2504, e.g., the beam spot centroid image data as shown in the examples of FIGs. 5B and 26; andWSGR Docket No. 64100-742.601 determining a number of imaged spots of the reflected beam that are present at a portion of the centroid image data at a corresponding z-stage position 2506 of the imaging apparatus. In some cases, the centroid image data, as shown in the examples of FIGs. 5B and 26, may be processed going from left to right. In some cases, the horizontal line 2644 of the centroid image data shown in the example of FIG. 26 may indicate and / or represent a dynamic range of the imaging apparatus, described herein, where centroid image data that is detected and / or identified above the horizontal line 2644 may be outside the dynamic detection range of the imaging apparatus.

[0119] In some embodiments, the method 2500 may further comprise processing the portion of the centroid image data to determine if one spot is determined and / or detected for the portion of the centroid data at the corresponding z-stage position. In some embodiments, the method 2500 may further comprise determining whether the one spot is below any two spot locations in the portion of the centroid image data 2508. In some embodiments, if the spot is below any two spot locations, then the spot may be determined and / or identified to be a spot of the beam of the light source reflected from a first surface 2510. In some embodiments, if the spot is not below any two spot locations, a determination may be made whether the second surface has been identified and / or determined from another process and / or operation 2512. In some embodiments, if the second surface has not been identified and / or determined from another process and / or operation, the spot may be determined and / or identified to be a spot of the reflected beam off of the first surface 2514. In some embodiments, if the second surface has been identified and / or determined from another operation and / or process, a determination may be made whether the spot belongs to a reflection of the beam of the light source from the first surface or the second surface 2516. In some embodiments, if the spot is identified and / or determined to not be a spot of a reflected beam of the light source off of the first or second surface, then the spot may be determined and / or identified to be a reflection the beam of the light source reflected off of a third surface 2518. In some embodiments, if the spot is identified and / or determined to be a spot of a reflected beam of the light source off of the first or the second surface, then the spot may be identified and / or determined as a spot of a beam of the light source reflected off of a first or a second surface 2520.

[0120] In some embodiments, the method 2500 may further comprise processing the portion of the centroid image data to determine if two spots are determined, detected, and / or identified for the portion of the centroid image data. In some embodiments, if two spots are determined, then a determination may be made whether more than four spots are generated from the beam of the light source reflecting off of the first surface have been identified by another process and / or operation 2522. In some embodiments, if more than four spots corresponding to the first surface have been identified by another process and / or operation, then a determination may be made whether the first spot of the four or more spots may be a spot of the beam of the light source reflected off ofWSGR Docket No. 64100-742.601 the first surface 2524. In some embodiments, if less than four spots of the first surface have been identified by another process and / or operation, then the method may return thatthere is insufficient data to classify which surface the spot corresponds to 2526. In some embodiments, if the first spot of the four or more spots is a spot of the beam of the light source reflected off of the first surface, then a determination may be made whether the second spot of the four or more spots is a spot generated by a beam of the light source reflected from the second surface 2530. In some embodiments, if the second spot of the four or more spots is a spot of a beam of the light source reflected from the second surface, then the second spot of the four or more spots may be identified as a spot of the beam of the light source reflected from the second surface 2534. In some embodiments, if the second spot of the four or more spots is not a spot of a beam of the light source reflected from the second surface, then the second spot may be identified as a spot of the beam of the light source reflected from the third surface 2532. In some embodiments, if the first spot of the four or more spots is not a spot of a beam of the light source reflected off of the first surface then a determination may be made whether the second spot of the four or more spots is a spot of the beam of the light source reflected off of the first surface 2528. In some embodiments, if the second spot of the four or more spots is not a spot of the beam of the light source reflected from the first surface, then the first spot of the four or more spots may be identified as a spot of a beam of the light source reflected off of the second surface and that the second spot may be identified as a spot of a beam of the light source reflected from the third surface 2536. In some embodiments, if the second spot of the four or more spots is the spot of the beam of the light source reflected from the first surface, then a determination may be made whether the first spot of the four or more spots may be a spot of abeam of the light source reflected from the second surface 2538. In some embodiments, if the first spot ofthefour or more spots is a spot of a reflected beam of the light source from the second surface, then the first spot may be identified as a spot of a beam of the light source reflected from the second surface 2540. In some embodiments, if the first spot of the four or more spots is not a spot of a reflected beam of the light source from the second surface, then the first spot of the four or more spots may be identified as a spot of a beam of the light source reflected from the third surface 2542.

[0121] In some embodiments, the method 2500 may further comprise processing the portion of the centroid image data to determine if three spots are determined and / or detected for the portion of the centroid image data at the corresponding z-stage position. In some embodiments, if three spots are determined and / or detected for the portion of the centroid image data, then a first, second, and third surface, may be determined and / or identified from the first, second, and third spot, respectively 2544.WSGR Docket No. 64100-742.601

[0122] In some cases, up to about 4 portions of the centroid image data across four z-positions of the z-stage 2640 may be used to process whether a beam spot centroid image at a fifth z-position of the z-stage 2642 may be determined and / or identified to be part of the one or more portions of the centroid image data of a surface of the one or more surfaces, as shown in the example of FIG. 26. For example, if a fifth z-position centroid image fits a linear fit of the up to four beam spot centroid image positions, the fifth z-position may be identified and / or determined to be spot of a beam of the light source reflected off of the surface identified by the up to four beam spot centroid images.Fluid Level Measurements

[0123] Accurate fluidic dispensing and aspiration can be important for the performance of open fluidic systems that are used for performing chemical reactions or molecular biological assays. Fluidic calibration and failure detection may originate from human intervention due to off- instrument volumetric measurements with external reagents and parts. The present disclosure provides methods or systems for an open fluidics on-instrument self -calibration system. The methods may leverage an imaging apparatus that comprises an autofocus (ATF) module comprising an autofocus sensor and an optical and light producing components in order to measure volume at a location, for example, volume of a fluid in a well in an open channel fluidic system that can perform self-calibration or detecting when a fluidic failure occurs during an assay. When used in microscopy, the ATF sensor may detect an interface between two mediums with different refractive indexes, for example a glass slide and a tissue or reagent on a slide, etc., to provide or collect an image of a biological sample or fluid disposed on a surface with optimal contrast.

[0124] Moreover, open fluidic systems can face many technical challenges. For example, in an open fluidic system it can be difficultto detect fluidic failures such as over dispensing of reagents, or not aspirating reagents, etc., before, during or post-run, where a run failure may be detected by a technician post-run. Late detection of a failed run may result in waste of reagents, money, or technician time or waste of a sample as the run may need to be repeated. The performance of fluidic pumps or aspirators may drift over time, requiring regular calibrations for maintaining robust and reliable instrument performance. Existing calibration methods require technical expertise to measure volume dispensed or aspirated and calibrate the device from those measurements. Additionally, volumetric measures require external parts and reagents that add another layer of cost to fluidic systems. Precision volumetric measuring kits for measuring volumes in open fluidic systems can be costly, and hand pipetting and running the measurements are prone to technician or calibration errors. As such, off instrument types of measurements and calibrations are not ideal.WSGR Docket No. 64100-742.601

[0125] Performing on-instrument self -calibration may provide a number of benefits over off- instrument calibration. For example, on-instrument calibration may eliminate the reagents and devices for practicing off-instrument calibration. As there is no addition of off -in strument reagents, the risk of contamination or potential clogging of fluidics lines by off -instrument reagents may be eliminated. Additionally, use of on-instrument reagents may represent the solutions used for an assay that differ from reagents that are added for off-instrument calibration. Moreover, technical errors based on human off-instrument calibration are eliminated when selfcalibration is done on-instrument. As the calibration system is on-instrument, it may be possible to run the calibration at different times (e.g., one or more times through the course of imaging one or more surfaces and one or more biological samples or fluids disposed thereto) by programming theinstrumentto run the self -calibration. By programingtheinstrumentto run self-calib ration one may potentially catch an instance where, e.g., there is an issue with fluid volumes in a well such that an assay may be adversely affected. In this example, the assay may be stopped early, thereby saving reagents, time, and cost of running and re-running the assay if an issue is encountered. Accordingly, there is a lack of practical or economical solutions to measure the volume of the liquid dispensed or aspirated, making fluidic systems, e.g., an open fluidic system prone to variations or failures.

[0126] The present disclosure utilizes the system or methods described herein, for the use of on- instrument fluidic self-calibration in an open fluidic system. The present disclosure may utilize the ATF sensor to detect different refractive indexes of a substrate, a sample disposed thereon, or an environment surrounding the substrate or the sample. For example, methods disclosed herein may translate a z-stage operational movement of the ATF module coupled thereto, to detect or determine a position of (1) the bottom of the substrate, (2) the interface between the substrate and an aqueous solution, or (3) the interface between the aqueous solution and the air above the aqueous solution. The autofocus sensor of the autofocus module may detect an interface between two mediums with different refractive indexes. When used in microscopy, the autofocus sensor may be used to, e.g., find the imaging surface between a glass slide and a component on a slide, such as a tissue, cells, a plurality of nucleic acids, or a dispensed liquid. The autofocus module may focus an image of the biological sample or fluid onto one or more detectors by an objective lens, described herein, that can travel in a focal z height with respect to a surface (e.g., of a microtiter plate) to provide an image of abiological sample or fluid disposed thereto, with the best contrast or resolution.

[0127] In some cases, the autofocus sensor may capture one ormore images atthethree interfaces. In some embodiments, the image data can be used to calculate the distance between the S2 and S3 interfaces, thereby determining the height of the liquid in a well. In some embodiments, theWSGR Docket No. 64100-742.601 zero position with zero crossing point in the sensor values may indicate an interface with two different refractive indexes. In some embodiments, the height of the liquid can be calculated by subtracting the z positions corresponding to the S2 and S3 interfaces.

[0128] In some instances, a standard curve representing the height of the liquid in a well and the corresponding volume of the liquid may be generated. In some cases, a standard curve may be generated by aliquoting volumes of liquid into the platform of interest, for example, into wells of a 96 or 384 well plate. In some embodiments, the standard curve liquids can then be assayed by the self -calibration system and the imaging data can be used to determine the height of the liquid that was aliquoted into the wells and graphs generated to demonstrate the relationship. In some cases, the fluid in the wells may be measured by units of microliters per micrometer (pL / pm).

[0129] To address the aforementioned shortcomings or challenges of open fluidic systems, the disclosure provides or describes a method 2300 (as shown in the example of FIG. 23) for determining a fluid level in a well, comprising: (a) directing a light beam from a light source to a portion of a surface and a third surface or interface of a well positioned proximate to a plurality of positions from a detector, where a fluid is disposed in the well between the surface and the third surface 2302; (b) receiving a reflected portion of the light beam from the second surface and the third surface or interface onto the detector at each position of the plurality of positions 2304; (c) identifyingafirstposition of the second surface andathird position of the third surface orinterface from the reflected portion of the light beam at each position of the plurality of positions 2306; and (d) determiningthe fluid level in the well from the first position of the second surface andthe third interface of the third surface 2308. In some embodiments, the well may comprise a first surface, where the first surface comprises a bottom surface of a microtiter plate or a multi-well plate. In some embodiments, the second surface may comprise a bottom surface of a well of a multi-well plate or a microtiter plate. In some embodiments, the third surface or interface may comprise a top surface of the fluid disposed in the well above the second surface. In some embodiments, the first surface may be disposed or provided up to about 10 mm below the bottom surface of the well (e.g., below the second surface). In some embodiments, the third surface or interface may be disposed or provided up to about 10 mm above the second surface of the well. In some embodiments, the second surface and the third surface or interface may be variable in position relative to each other. In some embodiments, the first surface and the second surface, may be at a fixed position relative to a home position of the detector. In some embodiments, the method 2300 may be performed in atime comprising at most about 500 ms, at most about400 ms, atmost about 300 ms, at most about 200 ms, at most about 100 ms, or less. In some embodiments, the time may be about 200 ms. In some embodiments, the light beam may comprise EM wavelengths in (i) the NIR spectrum, (ii) the UV spectrum, (iii) the visible spectrum, or any combination thereof. InWSGR Docket No. 64100-742.601 some cases, the fluid may comprise a wash buffer, detection reagent, or a combination thereof. In some instances, the fluid level in the well maybe determined and / or the system may be calibrated before one or more imaging processes to image one or more biological samples disposed within the well and / or on a surface of the well. In some cases, the calibration of the method of determining a fluid level in the well may be completed and / or conducted once the system described herein is manufactured and / or onceauser of the systems receives and / or operates the manufactured system. In some instances, the user may calibrate the method of determining a fluid level in the well by providing and / or using a well plate and / or cartridge with pre -dispensed known liquid volumes in one or more vessels and / or one or more wells of the well plate and / or cartridge. In some cases, calibration of the method of determining a fluid level may be conducted: during manufacturing of the system, when the system is installed at a point of use of the user, during preventative maintenance visits by a technician at the point of use, before each imaging process and / or operation imagingthe one or more biological samples disposed within the well, after each imaging process and / or operation imagingthe one or more biological samples disposed within the well, or any combination thereof.

[0130] In some embodiments, the determination of the fluid level and / or fluid volume in a well, described herein, may be based on a distance between an identified position of the second surface and the third surface multiplied by the surface area of the well. In some cases, the fluid level may be calibrated. In some instances, the fluid level and / or fluid volume in the well may comprise a distance between the second surface and the third surface or interface multiplied by a calibration factor. In some cases, the calibration factor may be determined based on a relationship of the distance between the position of the identified second surface and third surface or interface, shown as delta Z 2645 in the example of FIG. 26, and the volume in the well determined by manually aliquoting known volumes of fluid in one or more wells.

[0131] In some embodiments, the second surface may comprise an array of one or more other second surface portions of the second surface. In some embodiments, the second surface may comprise one or more other second surface portions of a microtiter plate. In some embodiments, the second surface may comprise one or more other second surface portions of a 96 -well microtiter plate, a 384-well microtiter plate, or a 1536-well microtiter plate. In some embodiments, the portion of the second surface may be patterned as a shape comprising (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a geometric shape, or (v) any combination of (i) - (iv). In some embodiments, the portion of the surface may comprise a surface area of about 0.02 cm2, about 0.03 cm2, about 0.05 cm2, about 0.08 cm2, about 0.1 cm2, about 0.2 cm2, about 0.3 cm2, about 0.4 cm2, about 0.5 cm2, about 0.6 cm2, about 0.7 cm2, about 0.8 cm2, about 0.9 cm2, about 1 .0 cm2, or greater.WSGR Docket No. 64100-742.601

[0132] In some embodiments, the method 2300 may comprise determining (i) a local tilt, (ii) a global tilt, or (iii) a combination of (i) and (ii), of the surface from a plane of the detector with respect to a fluid aspirator. In some embodiments, determining the local tilt may comprise: (a) obtaining a centroid of an image of the reflected portion of the light beam for each of the plurality of positions of the portion of the surface; (b) fitting a planar surface to the plurality of positions; and (c) determining a slope of the planar surface thereby determining the local tilt. In some embodiments, determining the global tilt may comprise: (a) obtaining a centroid of an image of the reflected portion of the lightbeam for each ofthe plurality of positions at a plurality of portions across the surface; (b) fitting a quadratic surface to the plurality of positions at the plurality of portions of the surface; and (c) determining a slope of the quadratic surface thereby determining the global tilt.

[0133] In some embodiments, the method 2300 may comprise using a z-stage to position the detector at the plurality of positions. In some embodiments, the z-stage may be configured with a travel range of the plurality of positions from the detector comprising a range of up to about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm or about 12 mm. In some embodiments, the z-stage maybe configured with a capture range of the plurality of positions from the detector comprising a range of up to about 2 mm. In some embodiments, the z- stage may be configured with a depth of focus of the plurality of positions from the detector comprising a range of up to about 5 pm with an accuracy of about 10% of the depth of focus. In some embodiments, a distance between the first position and the third position of the plurality of positions for SI, S2 or S3 may comprise at most about 100 pm, at most about 90 pm, at most about 80 pm, at most about 70 pm, at most about 60 pm, at most about 50 pm, at most about 40 pm, at most about 30 pm, at most about 20 pm, or at most about 10 pm. In some embodiments, the distance may be about 40 pm. In some embodiments, the method 2300 may comprise: (i) moving the detector continuously at a rate between each position of the plurality of positions, (ii) moving the detector periodically at a period between each position of the plurality of positions, or (iii) a combination of (i) and (ii). In some embodiments, the method may comprise using the first position of the surface and the third position of the third surface to autofocus the detector on one or more particles positioned proximate to or immobilized to the portion of the surface. In some embodiments, the one or more particles may comprise amplification products coupled to fluorescently labeled polynucleotides. In some embodiments, the amplification products or the fluorescently labeled polynucleotides coupled thereto may be suspended in a buffer. In some embodiments, the coupling may comprise hybridization of the amplification products to the fluorescently labeled polynucleotides. In some embodiments, the hybridization may comprise covalent bonds. In some embodiments, the hybridization may comprise non-covalent bonds. InWSGR Docket No. 64100-742.601 some embodiments, non-covalent bonds may include van der Waals forces, ionic forces, or hydrogen bonding.Computer systems

[0134] The present disclosure describes a computer system 2400, shown in the example of FIG. 24, that may be electrically coupled to one or more components of the systems or devices described herein, to control or actuate one or more components of the systems or devices. In some cases, the computer system 2400 may implement methods of the disclosure, described herein. In some cases, the computer system 2400 can control or direct translation or movement of the imaging apparatus; analyze or processes one or more images of an emitted beam of the imaging apparatus reflected from one or more surfaces; direct or actuate the tip, tilt, or rotational motors, e.g., the theta X motor orthe theta Y motor, described herein, to adjust a slope or tip ortilt of the surface to permit or enable the imaging apparatus to focus on the one or more surfaces.

[0135] The computer system 2400 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device. The computer system 2400 may comprise a central processing unit (CPU, also “processor”, “computer processor”, or “one or more processors”, described herein) 2402, which can be a single core or multi core processor, or a plurality of processors for parallel processing The computer system 2400 may comprise memory or memory location 2410 (e.g., random-access memory, read-only memory, or flash memory), electronic storage unit 2404 (e.g., hard disk), communication interface 2406 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 2408, such as cache, other memory, data storage or electronic display adapters. The memory 2410, storage unit 2404, communication interface 2406 and peripheral devices 2408 may be in communication with the CPU 2402 through a communication bus (solid lines), such as a motherboard. The storage unit 2404 can be a data storage unit (or data repository) for storing data. The computer system 2400 can be operatively coupled to a computer network (“network”) 2416 with the aid of the communication interface 2406. The network 2416 canbethe Internet, an internet or extranet, or an intranet or extranetthatmay bein communication with the Internet. The network 2416, in some cases, may be a telecommunication or data network. The network 2416 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 2416, in some cases with the aid of the computer system 2400, can implement a peer-to-peer network, which may enable devices coupled to the computer system 2400 to behave as a client or a server.

[0136] The CPU 2402 can execute a sequence of machine-readable instructions, which can be embodied in a program or software, described herein. The instructions may be storedin a memory location, such as the memory (2404, 2410). The instructions can be directed to the CPU 2402,WSGR Docket No. 64100-742.601 which can subsequently program or otherwise configure the CPU 2402 to implement methods of the present disclosure, described herein. Examples of operations performed by the CPU 2402 can include fetch, decode, execute, and writeback.

[0137] The CPU 2402 can be part of a circuit, such as an integrated circuit. One or more other components of the system 2400 can be included in the circuit. In some cases, the circuit may be an application specific integrated circuit (ASIC).

[0138] The storage unit 2404 can store files, such as drivers, libraries and saved programs. The storage unit 2404 can store global tilt or local tilt of one or more regions across one or more surfaces, a determined shift in milliradians of a first axis (e.g., an x-axis) or a second axis (e.g, a y-axis) of a surface to reduce a tip or tilt of the surface to less than about 5 mrad with respect to an imaging plan of the imaging apparatus. The computer system 2400 in some cases can include one or more additional data storage units that are external to the computer system 2400, such as located on a remote server that is in communication with the computer system 2400 through an intranet or the Internet 2416.

[0139] The computer system 2400 can communicate with one or more remote computer systems through the network 2416. For instance, the computer system 2400 can communicate with a remote computer system of a user. The remote computer systems may comprise, for example, personal computers (e.g., portablePC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), personal digital assistants, or any combination thereof. The user can access the computer system 2400 via the network 2416.

[0140] Methods, as described herein, can be implemented by way of machine (e.g., computer processor) executable code (e.g., software or instructions) stored on an electronic storage location of the computer system 2400, such as, for example, on the memory 2410 or electronic storage unit 2404. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 2402. In some cases, the code can be retrieved from the storage unit 2404 and stored on the memory 2410 for ready access by the processor 2402. In some situations, the electronic storage unit 2404 can be precluded, and machine-executable instructions are stored on memory 2410.

[0141] The code can be pre-compiled or configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre -compiled or as- compiled fashion.

[0142] Aspects of the systems and methods provided herein, such as the computer system 2400, can be embodied in programming. Various aspects of the technology may be thought of asWSGR Docket No. 64100-742.601“products” or “articles of manufacture” in the form of machine (or processor) executable code or associated data that is carried on or embodied in a type of machine readable medium. Machineexecutable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flashmemory) or a hard disk. “ Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives andthe like, which may provide non -transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computerinto the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical or electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non -transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0143] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium . Non-volatile storage media include, f or example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be usedto implement databases. Volatile storage media may comprise dynamic memory, such as main memory of such a computer platform. Tangible transmission media may comprise coaxial cables, copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier -wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore may comprise, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code or data. Any of these forms of computer readable mediaWSGR Docket No. 64100-742.601 may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0144] The computer system 2400 can include orbe in communication with an electronic display 2412 that comprises a user interface (UI) 2414 for providing, for example, a display of local tiptilt of a surface or global tip -tilt of a surface. In some cases, the local tip -tilt of the surface or the global tip-tilt of the surface may be superimposed on an image of the surface, e.g., an image of one or more wells of a multi -well plate. Examples of UI’ s include, without limitation, a graphical user interface (GUI) of software di splayed on a display 2412 of the computer system or web -based user interface.

[0145] While various embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example. Numerous variations, changes, or substitutions may occur without departing from the present disclosure. It can be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.Terms and Definitions

[0146] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present disclosure belongs.

[0147] As used herein, the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “ or” unless otherwise stated.

[0148] As used herein, the term “about” in some cases refers to an amount that is approximately the stated amount.

[0149] As used herein, the term “about” refers to an amountthat is near the stated amount by 10%, 5%, or 1%, including increments therein.

[0150] As used herein, the term “about” in reference to a percentage refers to an amount that is greater or less the stated percentage by 10%, 5%, or 1%, including increments therein.

[0151] As used herein, the phrases “at least one,” “one or more,” and “or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0152] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example. It is not intended that the present disclosure be limited by the specific examples provided within the specification. While the present disclosure has been described with reference to the aforementioned specification, the descriptionsWSGR Docket No. 64100-742.601 and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions may occur without departing from the present disclosure. Furthermore, it shall be understood that all aspects of the present disclosure are not limited to the specific depictions, configurations, or relative proportions set forth herein which depend upon a variety of conditions and variables. It may be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is therefore contemplated that the present disclosure shall also cover any such alternatives, modifications, variations, or equivalents. Itisintendedthatthe following claims define the scope of the present disclosure and that systems, methods and structures within the scope of these claims and their equivalents be covered thereby.EXAMPLESExample 1-Autofocus spot classification by RANSAC implementation

[0153] In some instances, a spot classification algorithm may be problematic in incorrectly determining spots, and thus the interfaces for SI, S2 and / or S3. A spot classification algorithm may be based on state diagrams that may make assumptions that there is only one spot detected for each of the interfaces SI, S2 and S3. An engineering margin threshold may be employed, wherein the engineering margin is based on the number of bit shift, for example wherein the threshold for identifying a spot for interface classification can be determined by implementing the following equation:Threshold = 128 * engineering margin + black_level

[0154] In this example, the engineering margin allows for auserto setthe threshold independently of the pixel depth of the images taken, thereby allowing a user to set a threshold for detection of spots, for example identifying more spots or less spots in an autofocus image. In this example, a lower set threshold may be more likely to capture more noise that is not spots, whereas a higher set threshold may not capture all of the spots. As such, an engineering margin can be optimized to mitigate both extremes to find a “sweet spot” threshold for spot classification.

[0155] Experiments were performed to examine the application of RANSAC for spot classification for interfaces of SI , S2 and S3. A wash bottle of water is loaded in the buffer bay of the instrument. The water is pumped to reagent lines in the system and water is added through the various reagent lines into wells of an assay plate usingthe normal operating commands present in the software for an assay. For some dispensing, a diaphragm pump may dispense reagent and the amount of dispensed reagent may correlate with a pump stroke that is set by adjusting a screw associated with the diaphragm pump. In this example, for the diaphragm pump(s), water is dispensed by this mechanism based on determined calculated amounts as described herein.WSGR Docket No. 64100-742.601Briefly, water is dispensed by several pump strokes and the volume of water per stroke is calculated and the strokes are adjusted accordingly. For the non-diaphragm pumps, the reagent (in this instance water) is dispensed based on how long the pump is powered, for example a cyclic pump that continuously pumps for a specific time, such as milliseconds or seconds, etc., as specified by the operational software. A time for the pumping operation is set, for example 200ms of pumping, and the volume dispensed / unit of time calculated. The correct amounts are dispensed by turning the pumps ON and the software dispensing the volume of predetermined reagent as calculated into the wells. The wells are imaged, and data is analyzed.

[0156] In some embodiments, a spot image classification algorithm may have an autofocus imaging threshold, for example an engineering margin of 32, that might report one spot for each interface, resulting in less spots available for linear fittings thereby reducing sensitivity of autofocusing of the interfaces SI, S2 or S3. FIG. 27A shown an example of this issue, such that with an engineering margin of 32 the S2 interface is not classifying some spots (hashed circle). Conversely, an autofocus imaging threshold where an engineering margin of 16 can be implemented may fail to identify the correct interface due to multiple spots detected. For example, three spot images may be detected at the interface at SI with lower threshold than the spot image classification algorithm to properly classify the interface thereby being unable to make the correct classification. FIG. 27B shows an example of this issue, where the hashed circle shows the spots were detected at the SI interface but the interface spots are not correctly classified.

[0157] One option for mitigating spot classification issues includes employing a RANdom SAmple Consensus (RAN SAC) regressor algorithm which has the ability to choose random samples and fit a single line with generated data. RAN SAC algorithm does have several constraints such as the slope of a line is expected to be 1 ± 0.2, and the lines are expected to be separated by at least 750 pm in this example. The RAN SAC algorithm, as shown in the example of FIG. 28, is utilized to analyze spot image data by repeating operations of FIG. 29 until there are no remaining spots or until three interfaces, for SI, S2 and S3, are assigned.

[0158] FIG. 29 shows an example of the RANSAC algorithm implemented for autofocus spot classification of interfaces SI, S2 and S3. With an engineering margin of 16, RANSAC was able to correctly identify multiple spot images and classify the correct interfaces, as such the autofocus spot classification utilizing a RANSAC algorithm was successful and SI, S2 and S3 were successfully detected and correctly assigned.Example 2- Fluidic pump calibration for reagent dispensing and aspirating

[0159] A post-volumetric dispensing measurement workflow can provide fluidic pump calibration value suggestions such as what volume of a particular reagent is dispensed per cycle,WSGR Docket No. 64100-742.601 for example the volume wash reagent or dehybridization reagent that is dispensed per well per cycle. A post-volumetric measurement workflow can also be implemented to the post-run maintenance wash protocol to suggest calibration of what volumes of reagents to dispense into each well of an assay plate, for example the correct volume of decoding reagent to each well for a detection cycle. FIG. 30 shows an example of a post-run maintenance wash protocol, wherein a fluidic measurement workflow using instrument autofocus 3060 as shown in the example of BIG. 31 is inserted into the protocol. The wash run protocol is initiated with the priming of the pump tubing 3005 and the decoding reagent tubes being emptied 3010. The decoding reagent tubes are filled with water 3015 and purged by half 3020. The dehybridization tubing is washed with water 3030 as is the wash tubing 3035. The columns of the assay plate, in this example columns 2 and 4-9 are washed with water several times 3040. The nozzles are dipped in the reagents for the next cycle 3045. Depending on the capacity of a waste container, the waste container can be emptied at any pointin the protocol, in this example at operation 3025. Use of an autofocus operation 3060 is, in this example, implemented between the wash 3040 and dipping the nozzles for reagent dispensing 3045.

[0160] FIG. 31 shows an example of the post-run volumetric measurement workflow using autofocus 3060 which can be implemented within the protocol of the example of FIG. 30 to calibrate for reagent dispensing. In this example, operation 3060 is implemented after the well washing 3040 and prior to reloading of the reagent dispensing nozzles 3045. Workflow 3060 comprises, in this example, aspirating water by the reagent nozzles 3061 and measuring the aspirated volume 3062. The aspirated water is dispensed, for this example in 50 pL aliquots 3063, and the volume of the dispensed water is measured 3064. Fluid in wells of columns2 and 4-9 (as shown in the example of FIG. 30) are aspirated 3065 and the aspirated volumes for the wells of the columns are measured 3066. The fluid in the wells of columns 2 and 4-9 are filled with, for example, 150 pL aliquots of water 3067, the nozzles are dipped 3068 and the waste container emptied 3069. While the present example of the workflows in FIG. 30 and FIG.31 measures and dispenses water as the liquid, practical application may result in measuring and dispensing assay reagents into a well.

[0161] For fluid calibration suggestions including fluidic x and y theta (0) values, reagent dispensing volume per cycle and the nozzle aspiration Z height can be made based in part on the workflow measurements of FIG. 31. As an example, to calculate the volume of reagent to be dispensed per cycle, the dispensing volumes of the pumps can be measured and the volume divided by the number of cycles in order to calculate the volume for dispensing per cycle, also known as the pump calibration value.WSGR Docket No. 64100-742.601

[0162] In this example, to calculate the 9 x and 9 y values, or plate-tilt with respecttothe aspirator, the residual volumes in a well can be measured after aspiration on instrument and the volumes can be fitted to a plane using the RANSAC algorithm to determine 9 x and 9 y values. For example, residual well reagent volumes can be measured with an offset in 9 x and 9 y values and fitted to a plane using the RANSAC algorithm. The measurements can be repeated with offset values, for example -5, -2.5, 0, 2.5 and 5 mrad in 9 x and 9 y, respectively. The measured offset values in 9 x and 9 y (e.g., the x and y slopes from a fitted plane) can be plotted over the given offset values and fitted as demonstrated in the examples of FIGs. 32A-B, thereby demonstrating that a given offset in 9 y can be directly measuredby the slope of the fitted plane in y with a 1 : 1 ratio. In FIG. 32A, this example shows the coefficient for 9 x is close to zero, indicating thatthe offset in 9 y did not impact the tilt in 9 x. This is also seen for offsets in 9 x in the example of FIG. 32B, wherein the offset in 9 x did not impact the tilt in 9 y. As such, the residual volumes in a well can be used to calculate and correct the tilt in an aspirator for correcting for the fluidic theta values.

[0163] The Z height dispensing and / or aspirating nozzle calibration suggestions can also be implemented based on the measurements of the example of FIG. 32. For example, the Z height of a nozzle can be calculated as:Z offset = (aspiration_median-asp_target_vol) / vol_per_mmExample 3- Fluidic calibration value suggestion example

[0164] For demonstration purposes of pump calibration value suggestions, the post-run volumetric measurement workflow (FIG. 30 and FIG. 31) is performed on an instrument with uncalibrated fluidics to produce the following pump and aspirator calibration suggestions for adjustment:Theta x = -3.748 mrad Theta y = -4.895 mrad Wash reagent volume / cycle / well = -3.37 pL Dehybridization reagent volume / cycle / well = -3.31 pL Z height = 0.276 mm

[0165] In this example, the suggestions for changes in nozzle reagent aspiration are implemented. A nozzle aspirated water that had been pre-pipetted into wells of a 48-well plate. Light gray bars in the FIG. 33 show examples of aspiration volumes from the six columns (eight wells / column) from a plate pre-calibration. Agradientacrossthe columns as well as variation within the columns was high due to tilt in both x and y directions. The residual volumes were higher than target volumes (aspiration target volumes as indicated by the black dotted line). Dark gray bars in the FIG. 33 shows examples of aspiration volumes after implementation of the calibrationWSGR Docket No. 64100-742.601 suggestions from the autofocus volumetric measurement workflow, which reduced the gradient and variation significantly resulting in residual volumes close to the target volumes.

[0166] Suggestions for changes in nozzle wash reagent and hybridization reagent dispensing are also implemented in this example. FIG. 34 shows examples of wash and dehybridization nozzle dispensed water (as a proxy for wash reagent) into wells of a 48 -well plate measured pre and postcalibration by autofocus. In FIG. 34, without calibration the dispensed volumes from the wash tubing are well below what is set for the lower level for wash volume dispensing as shown in the dark boxes. Following calibration, light grey boxes showed a marked improvement for wash and dehybridization reagent dispensing, with the majority of the dispensing volumes being between the upper and lower levels which may be expected for an assay.

[0167] While a 48-well plate is used in this example, a 96-well plate can also be utilized, or any other plate structure that is compatible with an assay instrument.

Claims

WSGR Docket No. 64100-742.601CLAIMSWHAT IS CLAIMED IS:

1. A system for calibrating instrument imaging apparatuses, the system comprising one or more processors and a memory storing one or more programs for execution by the one or more processors, the one or more programs comprising instructions to:(i) directa first light beam from a first light source of a first imaging apparatus to a portion of a surface positioned proximate to a plurality of positions on the surface, wherein a distance between the surface and the first imaging apparatus can change by translating an objective over the plurality of positions thereby generating a plurality of objective positions;(ii) receive a reflected portion of the first light beam onto an imaging sensor of the first imaging apparatus to form an image at each position of the plurality of objective positions;(iii) calculate a centroid sensor coordinate and size metric of the reflected portion of the first light beam for each position of the plurality of objective positions identified within the image of the first imaging apparatus;(iv) fit a linear portion of a relationship between the centroid sensor coordinates of the image from the first imaging apparatus over the plurality of positions and an objective translator position for a subset of the plurality of positions;(v) direct a second light beam from a second light source to a field of view of a second imaging apparatus and receive a reflected portion of the second light beam onto at least one imaging sensor of the second imaging apparatus to form a second image at each position of the plurality of positions, thereby generating a second set of images from a second set of imaging sensors of the second imaging apparatus over the subset of the positions of the plurality of positions by translating the objective on a z axis;(vi) compute a focus metric from the second set of images from the second imaging apparatus over the subset of the positions of the plurality of positions; and(vii) generate a focus model based at least on an optimized focus metric from the second imaging apparatus and the reflected beam centroid sensor coordinates from the first imaging apparatus, thereby calibrating the imaging apparatuses.

2. The system of claim 1 , wherein the instructions further comprise autofocusing an instrument after calibrating the instrument imaging apparatuses, wherein autofocusing comprises:(viii) translating the surface in one or more of an x-axis and a-y axis to a different position of the surface, and(ix) translating the objective to a position to receive a second reflected portion of the first light beam onto an imaging sensor of the first imaging apparatus, wherein centroid sensorWSGR Docket No. 64100-742.601 coordinates of the second reflected portion of the first light beam match a previously optimized centroid sensor coordinate from the first imaging apparatus.

3. The system of claim 1 or 2, wherein the first light beam is non -fluorescent and the second light beam is fluorescent.

4. The system of any one of claims 1-3, wherein the surface comprises a first surface and is located on a bottom of a multi -welled plate, and wherein the instructions further comprise:(a) performing (i) - (vii) for a position on a second surface, wherein the second surface comprises a bottom of a well of the multi-welled plate;(b) performing (i) - (vii) for a position on a third surface, wherein the third surface comprises a top of a fluid of a fluid / air interface located above the second surface; and(c) updating a linear portion of the second surface.

5. The system of claim 4, wherein the first surface is between about 0.1 millimeters and about 1.5 millimeters below the second surface, and wherein the third surface is about 2 millimeters to about 10 millimeters above the second surface.

6. The system of claim 4, wherein the position of the third surface is changeable relative to the position of the first surface or the position of the second surface, and wherein the difference between the second surface and the third surface indicates a level of the fluid in the well of the multi -welled plate.

7. The system of any one of claims 1-6, wherein fitting the linear portion of the relationship comprises:(a) using a function to determine a size metric for each image captured at each position of the plurality of objective positions corresponding to each centroid;(b) selecting the size metric which maximizes or minimizes the function; and(c) selecting a subset of centroids corresponding to (i) the selected size metric and (ii) size metrics between a first position and a second position of the plurality of positions, wherein each size metric is associated with a spot size of each image, and wherein the function comprises an exponential or parabolic function of the spot sizes of the subset of the centroids.

8. The system of any one of claims 1-7, wherein optimizing the focus metric comprises:(a) using a function to determine a focus metric for each image captured at each position of the plurality of objective positions corresponding to each centroid of the linear portion of the relationship; and(b) selecting the focus metric which maximizes or minimizes the function,WSGR Docket No. 64100-742.601 wherein each focus metric is associated with a spot size of each image, and wherein the function comprises (i) a full width at half maximum (FWHM) function, (ii) a Brenner gradient function, or (iii) a Shannon Entropy of Laplacian Pyramid (SLaP) function.

9. The system of any one of claims 1 -8, wherein the instructions further comprise determining (i) a local tilt, (ii) a global tilt, or (iii) a combination of (i) and (ii), of the surface from a plane of the imaging apparatuses.

10. The system of claim 9, wherein determining the local tilt comprises:(a) obtaining a centroid for each of the plurality of positions of the surface;(b) fitting a planar surface to the plurality of positions; and(c) determining a slope of the planar surface thereby determining the local tilt.

11. The system of claim 9, wherein determining the global tilt comprises:(a) obtaining a centroid for each of the plurality of positions of the surface;(b) fitting a parametric function to the plurality of positions of the surface; and(c) determining a slope of the parametric function thereby determining the global tilt.

12. The system of any one of claims 1-11, wherein the instructions are performed in a time of about 500 milliseconds, about 400 milliseconds, about 300 milliseconds, about 200 milliseconds, about 100 milliseconds, or less.

13. The system of any one of claims 1-12, wherein the instructions comprise using a stage to position the surface for the imaging apparatuses.

14. The system of any one of claims 1-12, wherein the instructions comprise positioning the objective in a z axis relative to the surface for the imaging apparatuses.

15. The system of claim 13, wherein the stage is configured with a travel range between the plurality of positions of up to about 6 millimeters.

16. The system of claim 13, wherein the stage is configured with a capture range of the plurality of positions of up to about 2 millimeters by the first imaging apparatus, the second imaging apparatus, or a combination thereof.

17. The system of claim 13, wherein the stage is configured with a depth of focus of the plurality of positions comprising a range of up to about 5 micrometers with an accuracy of about 10% of the depth of focus from the first image apparatus, the second imaging apparatus, or a combination thereof.

18. The system of any one of claims 1-17, wherein a distance between a first position and a second position of the plurality of positions is from about 5 nanometers to about 5 millimeters.

19. The system of any one of claims 1-17, wherein a distance between a first position and a second position of the plurality of positions is from about 100 nanometers to about 1 millimeter.WSGR Docket No. 64100-742.60120. The system of any one of claims 1-17, wherein a distance between a first position and a second position of the plurality of positions is approximately 40 micrometers.21 . The system of any one of claims 1-20, wherein the instructions further comprise (i) moving the first imaging apparatus, the second imaging apparatus, or a combination thereof continuously between each position of the plurality of positions at a rate, (ii) moving the imaging apparatus periodically between each position of the plurality of positions, or (iii) a combination of (i) and (ii).

22. The system of any one of claims 1-21, wherein the instructions further comprise using the calibrated image apparatuses to identify one or more particles.

23. The system of claim 22, wherein the one or more particles comprise amplification products hybridized to fluorescently labeled polynucleotides.

24. The system of claim 23, wherein the amplification products are immobilized on the second surface.

25. The system of any one of claims 1-24, wherein the second surface is the bottom of the well of the multi-welled plate selected from a 48-well multi-welled plate, a 96-well multi-welled plate, a 384-well multi-welled plate, or a 1 ,536-well multi-welled plate.

26. The system of claim 25, wherein the bottom of the well comprises a (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a v shape, (v) a geometric shape, or (vi) any combination of (i) - (v).

27. The system of claim 25, wherein the bottom of the well comprises a surface area of about 0.02 square centimeters (cm2), of about 0.03 cm2, of about 0.05 cm2, of about 0.08 cm2, of about 0.1 cm2, of about 0.2 cm2, of about 0.3 cm2, of about 0.4 cm2, of about 0.5 cm2, of about 0.6 cm2, of about 0.7 cm2, of about 0.8 cm2, of about 0.9 cm2, of about 1.0 cm2, or greater.

28. A method for calibrating imaging apparatuses, the method comprising:(a) directing a first light beam from a first light source of a first imaging apparatus to a position of a plurality of positions on a second surface;(b) receiving a reflected portion of the first light beam onto an imaging sensor of the first imaging apparatus to form a captured image for each position of the plurality of positions;(c) fitting a linear portion of a relationship between a centroid coordinate of the captured images from the first imaging apparatus at each position of the plurality of positions and an objective translator position for a subset of the plurality of positions;(d) directing a second light beam from a second light source to a field of view for a second imaging apparatus and receiving a signal from reflected light of the second light beam onto at least one imaging sensor of the second imaging apparatus to form a second captured image at each position of the plurality of positions;WSGR Docket No. 64100-742.601(e) generating the second captured image from a second set of imaging sensors of the second imaging apparatus over the subset of the plurality of positions by translating an objective on a z axis;(f) determining a focus metric from a set of captured images from the second imaging apparatus over the subset of the plurality of positions; and(g) generating a focus model based at least on an optimized focus metric from the second imaging apparatus and reflected beam centroid sensor coordinates from the first imaging apparatus, thereby calibrating the first imaging apparatus and the second imaging apparatus of an instrument.

29. The method of claim 28, further comprising autofocusing an instrument after calibrating the instrument imaging apparatuses, wherein the autofocusing comprises:(h) translating the second surface in one or more of an x-axis or a y-axis to a different position of the second surface, and(i) translating the objective to a position to receive a reflected portion of the light beam onto an imaging sensor of the first imaging apparatus, wherein the centroid sensor coordinates of the reflected portion of the light beam match a previously optimized centroid sensor coordinate.

30. The method of claim 28, wherein the first light beam of the first imaging apparatus is non- fluorescent and the second light beam of the second imaging apparatus is fluorescent.31 . The method of claim 29, further comprising repeating (a) - (d) for each of one or more other positions of the second surface.

32. The method of any one of claims 28-31, comprising:(i) performing (a) - (d) for a portion of a first surface, wherein the first surface is below the second surface;(ii) performing (a) - (d) for a portion of a third surface, wherein the third surface is above the second surface; and(iii) updating the linear portion of the second surface.

33. The method of claim 32, wherein the first surface is provided on a bottom of a multi -welled plate, wherein the second surface is provided on a bottom of a well of a multi -welled plate, and wherein the third surface is at a fluid / air interface provided in the well above the second surface.

34. The method of claim 33, wherein the first surface is about 0.1 millimeters to about 1.5 millimeters below the second surface, and wherein the third surface is about 2 millimeters to about 10 millimeters above the second surface.

35. The method of claim 32, wherein a difference between the second surface and the third surface indicates a level of fluid in the well of the multi -welled plate.WSGR Docket No. 64100-742.60136. The method of claim 28, wherein fitting the linear portion of the relationship comprises:(i) using a function to determine a size metric for each image corresponding to each centroid captured at each position of the plurality of positions;(i) selecting the size metric which maximizes or minimizes the function; and(i) selecting a subset of the centroids captured at each position of the plurality of positions corresponding to (A) the selected size metric and (B) size metrics determined between the first position and the second position of the plurality of positions, wherein each size metric is associated with a spot size of each captured image, and wherein the function comprises an exponential or parabolic function of combined spot sizes of the subset of the centroids.

37. The method of claim 28, wherein optimizing the focus metric comprises using a function to determine the focus metric, wherein the function comprises (i) a full width at half maximum (FWHM) function, (ii) a Brenner gradient function, or (iii) a Shannon Entropy of Laplacian Pyramid (SLaP) function.

38. The method of any one of claims 28-37, further comprising determining (A) a local tilt, (B) a global tilt, or (C) a combination of (A) and (B), of the second surface from a plane of as first imaging apparatus, a second imaging apparatus, or a combination thereof.

39. The method of claim 38, wherein determining the local tilt comprises:(a) obtaining a centroid for each of the plurality of positions of the second surface;(b) fitting a planar surface to the plurality of positions; and(c) determining a slope of the planar surface thereby determining the local tilt.

40. The method of claim 38, wherein determining the global tilt comprises:(a) obtaining a centroid for each of the plurality of positions of the second surface;(b) fitting a parametric surface to the plurality of positions of the surface; and(c) determining a slope of the parametric surface thereby determining the global tilt.41 . The method of any one of claims 28-40, further comprising using a stage to position the plurality of positions with respect to the first imaging apparatus, the second imaging apparatus, or a combination thereof.

42. The method of any one of claims 28-40, further comprising positioning the objective in a z axis relative to the second surface for the first imaging apparatus, the second imaging apparatus, or a combination thereof.

43. The method of claim 41 , wherein the stage is configured with a travel range of the plurality of positions of up to about 6 millimeters from the first image apparatus, the second imaging apparatus, or a combination thereof.WSGR Docket No. 64100-742.60144. The method of claim 41, wherein the stage is configured with a capture range of the plurality of positions of up to about 2 millimeters from the first image apparatus, the second imaging apparatus, or a combination thereof.

45. The method of claim 41, wherein the stage is configured with a depth of focus of the plurality of positions of up to about 5 micrometers with an accuracy of about 10% of the depth of focus from the first image apparatus, the second imaging apparatus, or a combination thereof.

46. The method of claim 28, wherein a distance between a first position and a second position of the plurality of positions comprises about 100 micrometers (pm), about 90 pm, about 80 pm, about 70 pm, about 60 pm, about 50 pm, about 40 pm, about 30 pm, about 20 pm, or about 10 pm.

47. The method of any one of claims 28-46, further comprising (i) moving the first imaging apparatus, the second imaging apparatus, or a combination thereof continuously between each position of the plurality of positions at a rate, (ii) moving the first imaging apparatus, the second imaging apparatus, or a combination thereof periodically between each position of the plurality of positions, or (iii) a combination of (i) and (ii).

48. The method of any one of claims 28-47, further comprising using the first calibrated image apparatus and the second calibrated image apparatus to identify one or more particles immobilized on the second surface.

49. The method of claim 48, wherein the one or more particles comprise amplification products hybridized to fluorescently labeled polynucleotides.

50. The method of claim 28, wherein the second surface comprises one or more other surface positions of a well of a 48-well multi-welled plate, a 96-well multi-welled plate, a 384-well multi -welled plate, or a 1536-well multi -welled plate.51 . The method of claim 50, wherein a bottom of the well comprises (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a v-shape, (v) a geometric shape, or (vi) any combination of (i) - (v).

52. The method of claim 51, wherein the bottom of the well comprises a surface area of about 0.02 square centimeters (cm2), of about 0.03 cm2, of about 0.05 cm2, of about 0.08 cm2, of about 0.1 cm2, of about 0.2 cm2, of about 0.3 cm2, of about 0.4 cm2, of about 0.5 cm2, of about 0.6 cm2, of about 0.7 cm2, of about 0.8 cm2, of about 0.9 cm2, of about 1.0 cm2, or greater.

53. A method for determining a fluid level in a well, the method comprising:(a) directing a light beam from a light source of a calibrated imaging apparatus to a position on a second surface and a position on a third surface of a well positioned proximate to a plurality of positions from a sensor, wherein a fluid is disposed in the well above the second surface, and wherein a fluid / air interface in the well is the third surface;WSGR Docket No. 64100-742.601(b) receiving a portion of a reflected light beam from the second surface and a portion of the reflected light beam from the third surface onto the sensor at each position of the plurality of positions of the second surface and at each position of the plurality of positions of the third surface;(c) using the portion of the reflected light beam from the second surface and the portion of the reflected light beam from the third surface to determine a position of the second surface and a position of the third surface based on the plurality of positions; and(d) determining the fluid level in the well from the determined position of the second surface and the determined position of the third surface.

54. The method of claim 53, wherein the second surfaceis a bottom of a well of a multi-welled plate.

55. The method of claim 53, wherein the third surface is about 2 millimeters to about 10 millimeters above the second surface.

56. The method of claim 53, wherein the second surface and the third surface are variable in position relative to each other.

57. The method of claim 53, further comprising determining (i) a local tilt, (ii) a global tilt, or (iii) a combination of (i) and (ii), of the second surface from a plane of the sensor.

58. The method of claim 57, wherein determining the local tilt comprises:(a) obtaining a centroid of an image of the reflected portion of the light beam for each of the plurality of positions of the second surface;(b) fitting a planar surface to the plurality of positions; and(c) determining a slope of the planar surface thereby determining the local tilt.

59. The method of claim 57, wherein determining the global tilt comprises:(a) obtaining a centroid of an image of the reflected portion of the light beam for each of the plurality of positions of the second surface;(b) fitting a parametric surface to the plurality of positions of the second surface; and(c) determining a slope of the parametric surface thereby determining the global tilt.

60. The method of claim 53, further comprising using a stage to position the sensor at the plurality of positions.

61. The method of claim 53, further comprising positioning an objective of the calibrated imaging apparatuses in a z axis relative to the surface.

62. The method of claim 60, wherein the stage is configured with a travel range of the plurality of positions from the sensor of up to about 6 millimeters.

63. The method of claim 60, wherein the stage is configured with a capture range of the plurality of positions from the sensor of a range up to about 2 millimeters.WSGR Docket No. 64100-742.60164. The method of claim 60, wherein the stage is configured with a depth of focus of the plurality of positions from the sensor of a range of up to about 5 micrometers with an accuracy of about 10% of the depth of focus.

65. The method of any one of claims 53-64, wherein a distance between a first position and an additional position of the plurality of positions on the second surface comprises about 100 micrometers (pm), about 90 pm, about 80 pm, about 70 pm, about 60 pm, about 50 pm, about 40 pm, about 30 pm, about 20 pm, or about 10 pm.

66. The method of any one of claims 53-65, further comprising (i) moving the sensor continuously between each position of the plurality of positions at a rate, (ii) moving the sensor periodically between each position of the plurality of positions, or (iii) a combination of (i) and (ii).

67. The method of claim 66, further comprising focusing the sensor on one or more particles immobilized on the second surface.

68. The method of claim 67, wherein the one or more particles comprise amplification products hybridized to fluorescently labeled polynucleotides.

69. The method of any one of claims 53-68, wherein the second surface is the bottom of a well of a 48-well multi-welled plate, a 96-well multi-welled plate, a 384-well multi-welled plate, or a 1536-well multi-welled plate.

70. The method of claim 69, wherein the bottom of the well comprises (i) a circular shape, (ii) a square shape, (iii) a rectangular shape, (iv) a geometric shape, or (v) any combination of (i) - (iv).71 . The method of claim 70, wherein the bottom of the well comprises a surface area of about 0.02 square centimeters (cm2), of about 0.03 cm2, of about 0.05 cm2, of about 0.08 cm2, of about 0.1 cm2, of about 0.2 cm2, of about 0.3 cm2, of about 0.4 cm2, of about 0.5 cm2, of about 0.6 cm2, of about 0.7 cm2, of about 0.8 cm2, of about 0.9 cm2, of about 1.0 cm2, or greater.

72. The method of claim 53, wherein the determined fluid level in the well is used to detect a fluidic failure of a fluidic system.

73. An imaging apparatus, the apparatus comprising:(a) a light source configured to direct a light beam to a portion of a surface positioned proximate to a plurality of positions relative to an imaging apparatus;(b) a sensor configured to receive a reflected portion of the light beam onto the sensor of the imaging apparatus thereby forming an image at each position of the plurality of positions; and(c) an automatic focuser configured to:WSGR Docket No. 64100-742.601(i) fit a linear portion of a relationship to a centroid of each image between a first position and a second position of the plurality of positions;(ii) optimize a focus metric obtained from the linear portion of the relationship; and(iii) generate a focus model based at least on the optimized focus metric.

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