Instrument registration for multi-well plate

Abstract

The present disclosure provides methods, compositions and systems for registering a multi-well plate in an instrument. Registration of the location of wells in a multi-well plate may provide for accurate imaging of an assay substrate and accurate dispensing of assay reagents.

Description

WSGR Docket No. 64100-750.601INSTRUMENT REGISTRATION FOR MULTI-WELL PLATECROSS-REFERENCE

[0001] The present application claims the benefit of United States Provisional Patent Application Number 63 / 717,631, filed November 7, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Biological assays can be complex. Some biological assays require an instrument to detect an activity or response that is the result of a particular assay being used, for example, detecting a measurable light signature based on the presence of a target of interest. For an instrument to detect, for example, an event such as a measurable light signature from a biological reaction or a light signature from a detectable moiety that is introduced into an assay, the instrument may need to decipher where to look for that light signature.

[0003] Registration of an assay substrate by an instrument can be performed to orient instrument optics to where imaging may be required. Fiducials can be utilized to register an assay substrate by an instrument. Fiducials can include items such as, for example, raised areas on a substrate, etching on a substrate, fluorescent moieties immobilized on a substrate, or any other type of physical or chemical mechanisms whereby an instrument can register the placement of the fiducials for orienting the instrument as to where imaging may need to take place. For example, a multi-well plate can comprise various separate substrates, such as for example wells, for assay detection wherein the instrument may need to identify where imaging may take place for each of the separate substrates of the multi-well plate. A 96-well plate, for example, comprises 96 wells wherein each well of the 96 wells may require imaging to detect an outcome of an assay. The existing substrates in a multi-well plate may comprise small surface areas, and the physical or chemical manipulation needed for each well to be registered for imaging may not be possible. As such, there is a need for methods and compositions for registering multi-well assay formats by an imaging instrumentSUMMARY

[0004] 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 inWSGR Docket No. 64100-750.601 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.

[0005] Aspects disclosed herein provide multi-well plate configurations comprising: (a) a multi-well plate component comprising a plurality of wells; (b) a substrate component configured to receive biological samples; and (c) an autofluorescent pressure sensitive adhesive component, wherein the autofluorescent pressure sensitive adhesive component comprises a plurality of hole features positionally located between corners of adjacent wells of the plurality of wells. In some embodiments, the hole features of the plurality of hole features serve as fiducial markers. In some embodiments, the multi-well plate component is affixed superior to the autofluorescent pressure sensitive adhesive component and the substrate component is affixed inferior to the autofluorescent pressure sensitive adhesive component. In some embodiments, the substrate component comprises glass. In some embodiments, the glass is optically clear glass. In some embodiments, the autofluorescent pressure sensitive adhesive component autofluoresces between about 400 nm and about 650 nm. In some embodiments, the autofluorescent pressure sensitive adhesive component comprises a thermoplastic polymer carrier film. In some embodiments, the thermoplastic polymer carrier film comprises one or more of polyester, polypropylene, polyethylene terephthalate, or biaxially oriented polypropylene. In some embodiments, the thermoplastic polymer carrier film comprises an adhesive on a first side of the thermoplastic polymer carrier film and an adhesive on a second side of the thermoplastic polymer carrier film. In some embodiments, the adhesive comprises silicone. In some embodiments, the adhesive comprises acrylate. In some embodiments, the autofluorescent pressure sensitive adhesive component comprises a thickness of between about 48 pm and about 250 pm. In some embodiments, the hole features of the plurality of hole features comprise a diameter of about 0.5 mm to about 3 mm. In some embodiments, the hole features of the plurality of hole features are laser-cut into the autofluorescent pressure sensitive adhesive component. In some embodiments, the hole features of the plurality of hole features are die-cut into the autofluorescent pressure sensitive adhesive component. In some embodiments, the autofluorescent pressure sensitive adhesive component is transparent. In some embodiments, the autofluorescent pressure sensitive adhesive component is opaque.

[0006] Aspects disclosed herein provide methods, the method comprising: a) providing a multi-well plate comprising: i) a multi-well component comprising a plurality of wells, wherein the multi-well component is affixed superior to a pressure sensitive adhesive component and ii) a substrate component affixed inferior to the pressure sensitive adhesive component, wherein the pressure sensitive adhesive component comprises a plurality of fiducial markers; b) imaging the multi -well plate, wherein the imaging comprises capturing fluorescent images of the fiducialWSGR Docket No. 64100-750.601 markers of the plurality of fiducial markers, c) registering locations of the fiducial markers of the plurality of fiducial markers of the pressure sensitive adhesive component, and d) determining a location of one or more wells of the plurality of wells for imaging based on the registering of the locations of the fiducial markers. In some embodiments, the method further comprises determining a location for fluid dispensing in the one or more wells based at least in part on the registering of the locations of the fiducial markers. In some embodiments, the plurality of wells comprises between six and 1,536 wells. In some embodiments, the pressure sensitive adhesive component is double sided. In some embodiments, the pressure sensitive adhesive component autofluoresces between about 400 nm and about 650 nm. In some embodiments, the pressure sensitive adhesive component comprises a thermoplastic polymer carrier film. In some embodiments, the thermoplastic polymer carrier film comprises one or more of polyester, polypropylene, polyethylene terephthalate, or biaxially oriented polypropylene. In some embodiments, the pressure sensitive adhesive component comprises a thickness of between about 48 pm and about 250 pm. In some embodiments, the fiducial markers of the plurality of fiducial markers comprise hole features comprising a diameter of approximately about 0.5 mm to about 3 mm. In some embodiments, the thermoplastic polymer carrier film is transparent. In some embodiments, the thermoplastic polymer carrier film is opaque. In some embodiments, the method further comprises disposing a plurality of biological samples into one or more wells of the plurality of wells. In some embodiments, the biological samples of the plurality of biological samples comprise nucleic acids, proteins, or a combination thereof. In some embodiments, the fiducial markers of the plurality of fiducial markers are located equidistant between corners of adjacent wells of the plurality of wells of the multi -well plate component.

[0007] Aspects disclosed herein provide compositions for centering an imaging device on a well of a multi-well plate, comprising (a) a pressure sensitive adhesive, wherein the pressure sensitive adhesive is autofluorescent, and (b) a plurality of hole features positioned equidistant between comers of adjacent wells of the multi-well plate.

[0008] Aspects disclosed herein provide systems for registering one or more fiducial markers for a multi-well plate, comprising: a) a computer system comprising instructions for executing a centering program, wherein the centering program is configured to: (i) input a fluorescent image of the one or more fiducial markers from the multi -well plate; (ii) subtract a background from the fluorescent image of the one or more fiducial markers; (iii) determine a region of interest in the fluorescent image of the one or more fiducial markers; (iv) extract a foreground from the region of interest in the fluorescent image of the one or more fiducial markers; (v) circle fit the one or more fiducial markers; (vi) determine a radius of the one orWSGR Docket No. 64100-750.601 more fiducial markers; and (vii) determine an intersection of union of the one or more fiducial markers; and b) register the one or more fiducial markers based at least in part on (i)-(vii) of the centering programINCORPORATION BY REFERENCE

[0009] 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 the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] 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 drawing support.

[0011] FIGs. 1A-C show FIG. 1A) a first multi-well plate component of the present disclosure comprising 96 wells, FIG. IB) a first component of a multi-well plate of the present disclosure comprising pressure sensitive adhesive (PSA) with fiducial holes, and FIG. 1C) an example of a third component of a multi-well plate of the present disclosure comprising a glass bottom.

[0012] FIG. 2 shows an example of a drawing of a pressure sensitive adhesive layer of the present disclosure.

[0013] FIG. 3 shows an example of a bottom view of a multi-well plate comprising the combined components shown in FIGs. 1A-1C.

[0014] FIG. 4 shows examples of dimensions of the PSA holes used as fiducial markers as found when in a completed multi-well plate configuration.

[0015] FIGs. 5A-C show FIG. 5A) an isometric view of a top of a multi -well plate comprising fiducials as described herein, FIG. 5B) an example of a cross-sectional view of wells of a multi -well plate comprising fiducials as described herein, and FIG. 5C) an example of a side view of the layers of the multi-well plate comprising fiducials as described herein.WSGR Docket No. 64100-750.601

[0016] FIG. 6 shows an example of an analysis pipeline for identifying fiducials for use in downstream biological assays as described herein.

[0017] FIGs. 7A-D show FIG. 7A) an example of an image of a fiducial after applying an intersection of union calculation where the loU is <0.1 (poor), FIG. 7B) an example of an image of a fiducial after applying an intersection of union calculation where the loU is 0.1 -0.2 (fine), FIG. 7C) shows an example of an image of a fiducial after applying an intersection of union calculation where the loU is >0.2 (good), and FIG. 7D) an artificially generated image that represents the ground truth for FIGs. 7A-7D.

[0018] FIG. 8 shows examples of locations for fiducial investigation and data analysis as illustrated in the examples of FIGs. 9-11.

[0019] FIG. 9 shows examples of fiducial investigation data for the rows and columns identified in the example of FIG. 8.

[0020] FIG. 10 shows examples of additional fiducial investigation data for the rows and columns identified in the example of FIG. 8.

[0021] FIG. 11 shows examples of fiducial investigation data for row B and columns 2, 3, 4 and 5 identified in the example of FIG. 8.

[0022] FIG. 12 shows examples of grayscale images with fitted circles.

[0023] FIGs. 13A-B show examples of FIG. 13A) grayscale, foreground and background images and fitted circles taken in channel 1, and FIG. 13B) examples of grayscale, foreground and background images and fitted circles taken in channel 2.

[0024] FIGs. 14A-C show FIG. 14A) an example of a background image illustrating a fiducial marker background, FIG. 14B) an example of an image illustrating a fiducial marker when background is subtracted, and FIG. 14C) an example of an image illustrating a fiducial marker fitted circle after background subtraction.

[0025] FIG. 15 shows an example of a progression of images when practicing the pipeline of FIG. 6

[0026] FIG. 16 shows an example of pixel intensity of the fitted circle from the example of FIG. 14C

[0027] FIGs. 17A-B show FIG. 17A) an example of an autocentering pipeline for an autocentering registration algorithm, and FIG. 17B) an example of a position measurement illustration as part of the pipeline of FIG. 17A.

[0028] FIG. 18 shows an example workflow of how the autocentering registration model may be used in a XY Stage control system to move to any well location with micron level accuracy.WSGR Docket No. 64100-750.601DETAILED DESCRIPTION

[0029] The present disclosure provides robust methods to filter out poor results by increasing the confidence level of the fitting quality of optics systems. The present disclosure mitigates variations in sampling locations across a multi-well plate and determines the sample locations of a multi -well plate. The present disclosure provides for sub -micron level accuracy for determining the spatial location of wells of a multi-well plate autocentering by leveraging pressure sensitive adhesive (PSA) machined hole features as fiducial markers for determining the location of wells for accurate imaging and liquid dispensing.

[0030] There exists challenges in registering multiple substrates in biological assaying using multi-well plates. As such, there is a need for methods, systems, and compositions for registering multi-well assay formats by an imaging instrument.

[0031] Additional examples of assays canbe found in WO2022 / 109496, WO2023 / 096671, WO2023 / 096672, WO2023 / 096675, WO2023 / 096674, WO2025 / 212672, WO2025 / 019647, and WO2025 / 221697, each of which is incorporated herein by reference in its entirety.

[0032] There are advantages to performing autocentering in addition to accurate imaging and liquid dispensing. These advances include, but are not limited to, providing improved spatial awareness for the instrument reagent aspirating and dispensing nozzles so they do not impact any surface in a well of a multi-well plate. An additional advantage includes mitigating overexposure of the biological entities being assayed to light (e.g., photobleaching). An additional advantage includes maximizing the imaging area for reliably imaging enough biological entities to provide robust assay data yield. As such, disclosed herein are methods and compositions comprising a combination of processes and device designs that enable micron - level positioning accuracy of a spatial map of a multi-well plate leveraging machined hole features as fiducial markers for autocentering.

[0033] The present disclosure provides methods, compositions, and systems for locating features of multi-well plates for processing, imaging, and image data analysis. The methods, compositions and systems disclosed herein can be used to register features associated with a multi-well plate for determining locations for imaging during assays, such as assays comprising the analysis of biological samples. The methods, compositionsand systems disclosed herein can be used for determining locations for optimal reagent dispensing in a multi-well plate. As such, the present disclosure provides machined hole features in a pressure sensitive adhesive (PSA) which can be leveraged as fiducial configurations for allowing an instrument to correctly place imaging components for detecting the outcome of an assay, for example, for the analysis of biological samples. The fiducial configurations of the present disclosure may also be used to allow for accurate dispensing of reagents for an assay. As such, PSA hole features leveraged asWSGR Docket No. 64100-750.601 fiducials disclosed herein can enhance detection of an assay, enhance fluid dispensing of an assay, improve data processing, or the like.

[0034] The fiducials of the present disclosure provide for instrument registration for a multi well plate assay format. In some embodiments, fiducial markers, also known as “fiducials”, are located at known locations with respect to the substrates of a multi -well plate to assist in locating the substrates in the system for capturing events as part of an assay and for fluid dispensing and aspirating tasks such that fluids, via the location and identification of fiducials, can be dispensed accurately for a given sub strate of a multi-well plate (e.g., the bottom of a well of a multi-well plate). In some embodiments, the fiducials of the present disclosure leverage PSA hole features, thereby negating the need to tool or design additional fiducial markers into / onto a substrate of a multi -well plate.

[0035] A multi-well plate may be used in the methods, compositions, and systems disclosed herein. The multi-well plate includes a plate, such as an assay plate, that comprises a plurality of physically separated wells for performing biological assays. A plurality of physically separated wells may include, but is not limited to, a plate partitioned into six wells, eight wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells, and the like. The plurality of wells may comprise more than or equal to about 5 wells, about 10 wells, about 15 wells, about 20 wells, about 25 wells, about 30 wells, about 35 wells, about 40 wells, about 45 wells, about 50 wells, about 55 wells, about 60 wells, about 65 wells, about 70 wells, about 75 wells, about 80 wells, about 85 wells, about 90 wells, about 95 wells, about 100 wells, about 110 wells, about 120 wells, about 130 wells, about 140 wells, about 150 wells, about 160 wells, about 170 wells, about 180 wells, about 190 wells, about200 wells, about 210 wells, about 220 wells, about 230 wells, about 240 wells, about 250 wells, about 260 wells, about 270 wells, about 280 wells, about290 wells, about 300 wells, about 350 wells, about 400 wells, about 450 wells, about 500 wells, about 550 wells, about 600 wells, about 650 wells, about 700 wells, about 750 wells, about 800 wells, about 850 wells, about 900 wells, about 1000 wells, about 1100 wells, about 1200 wells, about 1300 wells, about 1400 wells, about 1500 wells, or about 1600 wells. The plurality of wells may comprise less than or equal to about 5 wells, about 10 wells, about 15 wells, about 20 wells, about 25 wells, about 30 wells, about 35 wells, about 40 wells, about 45 wells, about 50 wells, about 55 wells, about 60 wells, about 65 wells, about 70 wells, about 75 wells, about 80 wells, about 85 wells, about 90 wells, about 95 wells, about 100 wells, about 110 wells, about 120 wells, about 130 wells, about 140 wells, about 150 wells, about 160 wells, about 170 wells, about 180 wells, about 190 wells, about 200 wells, about 210 wells, about 220 wells, about 230 wells, about 240 wells, about 250 wells, about 260 wells, about 270 wells, about280 wells, about 290 wells, about 300 wells, about 350 wells, about 400 wells, about 450WSGR Docket No. 64100-750.601 wells, about 500 wells, about 550 wells, about 600 wells, about 650 wells, about 700 wells, about 750 wells, about 800 wells, about 850 wells, about 900 wells, about 1000 wells, about 1100 wells, about 1200 wells, about 1300 wells, about 1400 wells, about 1500 wells, or about 1600 wells. In some instances, a multi -well plate is a cassette. For example, a cassette can be a glass or plastic slide or other flat surfaced substrate upon which can be affixed a partitioned gasket which, when affixed to the substrate, can generate a number of discrete wells for running assays. The gasket can be fluid impermeable and may be reversibly affixed, such that the gasket can be removed if needed for practicing methods of an assay.

[0036] As used herein, the terms “centering”, “autocenter”, and “autocentering” refer to a method that utilizes physical features of known size, shape and location on a substrate of interest, for example a multi -well plate, to automatically align or center the substrate’s coordinate system to an instrument coordinate system.

[0037] As used herein, the terms “fiducial”, “fiducial configuration”, and “fiducial marker” refer to a distinguishable feature to be used as a point of reference for a substrate. The “fiducial”, “fiducial configuration, or “fiducial marker” may comprise sites upon which a biological assay may be performed, including, for example, imaging, aspirating, or dispensing of assay reagents. A fiducial can include, but is not limited to, a mark, an object, an etch, a cutout, a shape (e.g., geometric shape such as a circle, a square, a triangle, crosshair, etc.), an edge, an irregularity, a pit, a post, or a collection of different features at known locations that can be used as a reference. In some embodiments, the point of reference is detectable in an image at the point of reference and has an x- and y- coordinate in a plane on the object on which it is located. As well, in some embodiments, the point of reference can be specified in a z-plane that is orthogonal to the x-y plane. In some embodiments, one or more fiducials comprise one or more coordinates that are relative to one or more other fiducials. As disclosed herein, the machined hole features of the PSA are leveraged as fiducials, negating the need to incorporate additional fiducials onto a multi-well plate. As such, in some embodiments, fiducials can be features that are native to a substrate. Fiducials, in some embodiments, as described herein, can facilitate autocentering of the system optics for accurate imaging, aspirating, and dispensing of assay reagents.

[0038] The present disclosure provides for existing features to serve as fiducial markers, for example, the present disclosure provides for hole features that are incorporated into a component of a multi-well plate that serve as fiducial markers. The hole features serving as fiducial markers of the present disclosure may be circles which can be combined with a robust autocentering analysis pipeline. The combination may thereby render the need to add external fiducial markers to a system unnecessary. Additionally, the PSA, which may comprise the hole features that can serve as fiducial markers may be autofluorescent. The autofluorescent PSA may thereby negateWSGR Docket No. 64100-750.601 the need for additional light sources for the instrument in identifying and autocentering when using the PSA hole features as fiducial markers.

[0039] Imaging systems may be used in the methods, compositions, and systems disclosed herein. The imaging systems that may leverage the autocentering of fiducial markers and associated algorithmic pipelines and systems disclosed herein may be any imaging system that utilizes multi-well plates or other substrates that comprise features tooled into the substrates proper. The machined features, for example, the hole features as cut into a PSA used to affix a multi-well component to a glass component, can be leveraged as fiducial markers. Imaging systems that may practice the methods, compositions and systems described herein include, but are not limited to, imaging systems for detecting fluorescence, imaging systems for detecting chemiluminescence, imaging systems for imaging live cells, imaging systems for imaging transmitted light, imaging systems for colorimetric imaging, imaging systems used in spatial transcriptomics, imaging systems used for in situ based cell assays, fluorescence microscopylike imaging systems, phase contrast imaging systems, confocal imaging systems, computer assisted microscopy imaging systems, and the like. The imaging systems may include imaging systems for detecting fluorescence. The imaging systems may include imaging systems for detecting chemiluminescence. The imaging systems may include imaging systems for imaging live cells. The imaging systems may include imaging systems for imaging transmitted light. The imaging systems may include imaging systems for colorimetric imaging. The imaging systems may include imaging systems used in spatial transcriptomics. The imaging systems may include imaging systems used for in situ based cell assays. The imaging systems may include fluorescence microscopy -like imaging systems. The imaging systems may include phase contrast imaging systems. The imaging systems may include confocal imaging systems. The imaging systems may include computer assisted microscopy imaging systems. The present disclosure is not limited to any particular imaging system. Any imaging system where registration of a multi-well plate is desired may benefit from the methods, compositions, pipelines and systems of the present disclosure.

[0040] In the present disclosure, the fiducials comprise a plurality of circles that are cut into an adhesive sandwiched between a multi-well plate component and a glass substrate. The fiducial markers, described herein as circles, may be cut in a pressure sensitive adhesive (PSA). In some embodiments, the PSA may comprise two sides, for example, a top side and a bottom side. In some embodiments, the PSA may comprise a double-sided carrier format wherein a bulk carrier material is adhesively coated on both the top side and the bottom side of the PSA. In some embodiments, the PSA may comprise a double-sided format. In some embodiments, the PSA may comprise a first release liner that may be removable. In some embodiments, the PSAWSGR Docket No. 64100-750.601 may comprise a first adhesive composition. In some embodiments, the PSA may comprise a carrier film. In some embodiments, the PSA may comprise a second adhesive composition. In some embodiments, the PSA may comprise a second release liner that may be removable. For example, a PSA may comprise a double-sided format comprising, from a top side to a bottom side, a first release liner that may be removable, a first adhesive composition, a carrier film, a second adhesive composition and a second release liner that may be removable, or any combination thereof. In some embodiments, the carrier of the PSA comprises a thermoplastic polymer film. In some embodiments, the carrier of the PSA comprises a transparent polypropylene film. In some embodiments, the carrier of the PSA comprises an opaque polypropylene film. In some embodiments, the opaque polypropylene film is substantially opaque. In some embodiments, the PSA comprises a biaxially oriented polypropylene (BOPP) film. In some embodiments, a thermoplastic polymer carrier film of the PSA is coated on a top side and a bottom side with an adhesive. In some embodiments, the adhesive may be a silicone adhesive. In some embodiments, a thermoplastic polymer carrier film of the PSA is coated on a top side and a bottom side with an acrylate adhesive. In some embodiments, one or both sides of the PSA may comprise a release liner. In some embodiments, a release liner on one or both sides of a PSA comprises polyester. In some embodiments, a release liner comprises polyethylene terephthalate (PET).

[0041] In some embodiments, the PSA is thermal resistant. In some embodiments, the PSA is substantially thermal resistant. In some embodiments, the PSA may be chemical resistant. In some embodiments, the PSA is substantially chemical resistant. In some embodiments, the PSA may be resistantto one or more of alcohols, dimethylsulfoxide (DMSO), acids, organic solvents, and the like. In some embodiments, the PSA may be resistant or substantially resistant to alcohols. In some embodiments, the PSA may be resistant or substantially resistant to dimethylsulfoxide (DMSO). In some embodiments, the PSA may be resistant or substantially resistant to acids. In some embodiments, the PSA may be resistant or substantially resistant to organic solvents. In some embodiments, the PSA can withstand rapid thermal cycling. In some embodiments, the PSA is compatible with enzymatic reaction reagents. In some embodiments, the top and bottom release liners are removed for non-removably affixing a multi-well plate component to one side of the PSA and a glass substrate to the other side of the PSA, wherein the PSA comprises multiple hole features which can serve as fiducial markers as described herein.

[0042] In some embodiments, the PSA comprises a thickness. The PSA may comprise a thickness of between about 48 micrometers (pm) and about 250 pm. In some embodiments, the PSA comprises a thickness of between about 100 pm and about 200 pm. In some embodiments, the PSA comprises a thickness of between about 125 pm and about 175 pm. In someWSGR Docket No. 64100-750.601 embodiments, the PSA comprises a thickness of between about 140 pm and about 150 pm. In some embodiments, the PSA comprises a thickness of more than or equal to about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 105 pm, about 110 pm, about 115 pm, about 120 pm, about 125 pm, about 130 pm, about 135 pm, about 140 pm, about 145 pm, about 150 pm, about 155 pm, about 160 pm, about 165 pm, about 170 pm, about 175 pm, about 180 pm, about 185 pm, about 190 pm, about 195 pm, about 200 pm, about 205 pm, about 210 pm, about 215 pm, about 220 pm, about 225 pm, about 230 pm, about 235 pm, about 240 pm, about 245 pm, about 250 pm, about 255 pm, about 260 pm, about 265 pm, about 270 pm, or about 275 pm. In some embodiments, the PSA comprises a thickness of less than or equal to about 275 pm, about270 pm, about 265 pm, about 260 pm, about 255 pm, about 250 pm, about 245 pm, about 240 pm, about 235 pm, about 230 pm, about 225 pm, about 220 pm, about 215 pm, about 210 pm, about 205 pm, about 200 pm, about 195 pm, about 190 pm, about 185 pm, about 180 pm, about 175 pm, about 170 pm, about 165 pm, about 160 pm, about 155 pm, about 150 pm, about 145 pm, about 140 pm, about 135 pm, about 130 pm, about 125 pm, about 120 pm, about 115 pm, about 110 pm, about 105 pm, about 100 pm, about 95 pm, about 90 pm, about 85 pm, about 80 pm, about 75 pm, about 70 pm, about 65 pm, about 60 pm, about 55 pm, about 50 pm, about45 pm, or about40 pm. In some embodiments, a uniformity of the PSA is between ±2 pm. In some embodiments, the uniformity of the PSA allows for differential adhesion. In some embodiments, the uniformity of the PSA is about 1.2 pm, about 1.4 pm, about 1.6 pm, about 1.8 pm, about 2.0 pm, about 2.2 pm, about 2.4 pm, about 2.6 pm, about 2.8 pm, about 3.0 pm, about 3.2 pm, about 3.4 pm, about 3.6 pm, about 3.8 pm, about 4.0 pm, about 4.2 pm, about 4.4 pm, about 4.6 pm, about 4.8 pm, or about 5.0 pm.

[0043] In some embodiments, the PSA is opaque. In some embodiments, the PSA is substantially opaque. In some embodiments, the PSA is black. In some embodiments, the PSA is substantially black. In some embodiments, the PSA comprises a polyester film. In some embodiments, the PSA comprises a transparent polyester film. In some embodiments, the PSA comprises a substantially transparent polyester film. In some embodiments, the PSA comprises a transparent polypropylene film. In some embodiments, the PSA comprises a sub stantially transparent polypropylene film. In some embodiments, the PSA comprises a black PET film. In some embodiments, the PSA comprises a substantially black PET film. In some embodiments, the PSA comprises a transparent PET film. In some embodiments, the PSA comprises a substantially transparent PET film. In some embodiments, both sides (e.g., a top side and a bottom side) of a PSA comprising a PET film are coated with an adhesive. In some embodiments, the adhesive on both sides (e.g., a top side and a bottom side) of the PET film of aWSGR Docket No. 64100-750.601PSA comprise a release liner. In some embodiments, the top side of the PET film of a PSA comprises a release liner. In some embodiments, the bottom side of the PET film of a PSA comprises a release liner. In some embodiments, the release liner comprises PET. In some embodiments, the release liner comprises a polycoated kraft paper.

[0044] In some embodiments, the fiducial markers are cut into the PSA. In some embodiments, the fiducial markers are laser-cut into the PSA. In some embodiments, the fiducial markers are die-cut into the PSA. In some embodiments, the fiducial markers comprise autofluorescent properties. As such, the fiducial markers are visible upon imaging based on their autofluorescent properties and are not dependent on an accessory light source for illumination. In some embodiments, the autofluorescence of the fiducial markers is between about 400 nm and about 650 nm. In some embodiments, the autofluorescence of the fiducial markers is more than or equal to 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, or 750 nm. In some embodiments, the autofluorescence of the fiducial markers is less than or equal to 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, or 750 nm.

[0045] In some embodiments, a plurality of separate hole features can be leveraged as fiducial markers and the hole features may be cut into a PSA for autocentering and fiducial registration as described herein. In some embodiments, a minimum of three-hole features can be leveraged as fiducial markers and may be used for autocentering and fiducial marker registration. In some embodiments, more than or equal to about 3 hole features, 4 hole features, 5 hole features, 6 hole features, 7 hole features, 8 hole features, 9 hole features, 10 hole features, 15 hole features, 20 hole features, 25 hole features, 30 hole features, 35 hole features, 40 hole features, 45 hole features, 50 hole features, 55 hole features, 60 hole features, 65 hole features, 70 hole features, 75 hole features, 80 hole features, 85 hole features, 90 hole features, 95 hole features, 100 hole features, or more may be used in the methods, compositions, and systems disclosed herein. In some embodiments, less than or equal to about 3 hole features, 4 hole features, 5 hole features, 6 hole features, 7 hole features, 8 hole features, 9 hole features, 10 hole features, 15 hole features, 20 hole features, 25 hole features, 30 hole features, 35 hole features, 40 hole features, 45 hole features, 50 hole features, 55 hole features, 60 hole features, 65 hole features, 70 hole features, 75 hole features, 80 hole features, 85 hole features, 90 hole features, 95 hole features, 100 hole features, or more may be used in the methods, compositions, and systems disclosed herein.

[0046] FIGs. 1A-1C illustrates an example comprising a multi -well plate comprising hole features used as fiducials. FIGs. 1A-1C illustrates three main components. As illustrated in theWSGR Docket No. 64100-750.601 example of FIG. 1 A, the first component comprises a muti-well plate component, in this example, a 96 well plate. In some embodiments, the multi -well plate component of FIG. 1 A can be molded from one or more materials of polystyrene, polypropylene, cyclic olefin copolymer, acrylonitrile-butadiene-styrene, polyethylene, polyamide, polymethyl methacrylate, polycarbonate, or any combination thereof. In some embodiments, the multi-well plate component comprises polystyrene. In some embodiments, the multi-well plate component comprises polypropylene. In some embodiments, the multi-well plate component comprises cyclic olefin copolymer. In some embodiments, the multi-well plate component comprises acrylonitrile-butadiene-styrene. In some embodiments, the multi-well plate component comprises polyethylene. In some embodiments, the multi-well plate component comprises polyamide. In some embodiments, the multi-well plate component comprises polymethyl methacrylate. In some embodiments, the multi-well plate component comprises polycarbonate. In some embodiments, the multi -well plate is generated by injection molding. In some embodiments, the multi-well plate is generated by vacuum forming. In some embodiments, the multi-well plate is generated by computer numerical control machining. In some embodiments, the multi -well plate is generated by 3D printing. In some embodiments, the multi -well plate comprises a color. In some embodiments, the multi -well plate is black or substantially black, or another dark color that inhibits light contamination well to well. In some embodiments, the multi-well plate is opaque or substantially opaque.

[0047] The multi-well plate of FIGs. 1 A-1C may include a second component as illustrated in FIG. IB. In some embodiments, the second component comprises a pressure sensitive adhesive comprising hole features which may be leveraged for plate registration as fiducial markers. In some embodiments, the pressure sensitive adhesive comprises a double coated adhesive. In some embodiments, the double coated adhesive comprises a polyester carrier film. In some embodiments, the double coated adhesive comprises a polypropylene carrier film. In some embodiments, the carrier film is opaque or substantially opaque. In some embodiments, the carrier film is transparent or substantially transparent. In some embodiments, the adhesive comprises a material of one or more of acrylate, silicone, natural rubber, synthetic rubber, or the like. In some embodiments, the pressure sensitive adhesive is temperature resistant or substantially temperature resistant. In some embodiments, the pressure sensitive adhesive is chemical resistant or substantially chemical resistant. In some embodiments, the pressure sensitive adhesive is cut to fit a multi-well plate such that the pressure sensitive adhesive comprises cut-outs for each well of a multi -well plate in addition to hole features which serve as fiducial markers.WSGR Docket No. 64100-750.601

[0048] The example multi -well plate of FIGs. 1A-1C may include a third component illustrated in FIG. 1C, which comprises a glass substrate. In some embodiments, the glass substrate is an optically transparent glass substrate. In some embodiments, the glass substrate is a substantially optically transparent glass substrate. In some embodiments, the glass sub strate may comprise a refractive index. In some embodiments, the refractive index may be about 1.4 to about 1.7. In some embodiments, the optical glass may comprise an Abbe number. In some embodiments, the Abbe number of the optical glass is about 40 to about 60. In some embodiments, the glass substrate may be used as a substrate for biological assays. In some embodiments, the glass substrate comprises a surface coating or treatment. In some embodiments, the surface coating or treatment of the glass substrate can immobilize biological analytes. In some embodiments, biological analytes which can be immobilized on the glass substrate, include, but are not limited to, one or more of nucleic acids, proteins, cells, or tissues, or a combination thereof, which may be used in biological assays. In some embodiments, the surface coating or treatment on the glass substrate comprises one or more of an application of a cationic polymer, a gel, a charged coating, a neutral coating, a cationic polymer coating, a metallic coating, or a combination thereof. In some embodiments, the surface coating or treatment on the glass substrate comprises an application of a cationic polymer. In some embodiments, the surface coating or treatment on the glass substrate comprises an application of a gel. In some embodiments, the surface coating or treatment on the glass substrate comprises an application of a charged coating. In some embodiments, the surface coating or treatment on the glass substrate comprises an application of a neutral coating. In some embodiments, the surface coating or treatment on the glass substrate comprises an application of a cationic polymer coating. In some embodiments, the surface coating or treatment on the glass substrate comprises an application of a metallic coating.

[0049] Still referring to the examples illustrated in FIGs. 1A-1C, in some embodiments, a pressure sensitive adhesive (FIG. IB) is affixed to a multi-well plate component (FIG. 1A) prior to affixing the PSA (FIG. IB) to a glass substrate (FIG. 1C). For example, in some embodiments, the PSA (FIG. IB) is first affixed to the multi-well plate component (FIG. 1 A). In some embodiments, a release liner covers the PSA on the side opposite the PSA that is affixed to the multi -well plate component. In some embodiments, when the multi -well plate and affixed PSA are ready for affixing to the glass substrate, the release liner is removed from the PSA, the glass substrate is positioned on the PSA, and the glass substrate and the PSA may be affixed together by applying a pressure. In some embodiments, the glass substrate may be adhered to the PSA by lamination. In some embodiments, the glass substrate may be adhered to the PSA by bonding the glass substrate to the PSA.WSGR Docket No. 64100-750.601

[0050] In some embodiments, the PSA is aligned with the multi -well plate component and affixed to the bottom of the multi -well plate. For example, the PSA may comprise a release liner which can be manually removed, thereby exposing the adhesive of the PSA. The PSA and the multi-well plate can be aligned, for example manually or via an alignment tool or fixture that is configured to align two substrates within an alignment tolerance. In some embodiments, the PSA and the multi-well plate can be aligned via an alignment fixture or tool. In some embodiments, once the PSA is affixed to the multi -well plate via a first side of an adhesive, the glass substrate can be affixed to a second side of the adhesive of the PSA. For example, the second release liner can be removed from the PSA and the glass substrate can be affixed, either manually or via an alignment tool or fixture, to the second side of the adhesive of the PSA. In some embodiments, the affixing of the PSA to the glass substrate may result in incomplete bonding, voids or air gaps between the PSA and the glass substrate. In some embodiments, to remedy any incomplete bonding, voids or air gaps that may exist, an elastomeric gasket may be placed on the glass substrate which may be affixed to the multi -well plate through the PSA. In some embodiments, the gasketed multi -well plate can be placed into a bonding instrument, for example a laminating instrument, which comprises a pneumatic press, such that the bonding instrument can be used to apply force to the gasket and affixed multi-well plate. In some embodiments, the bonding instrument may be configured to provide vacuum pressure. In some embodiments, a manual roller or instrument roller can be used to provide pressure to bond the PSA to the glass substrate. In some embodiments, once the applied force process is complete, the multi-well plate can be removed from the instrument, and the gasket can further be removed from the multi-well plate. In some embodiments, a bond quality can be reassessed to determine whether the multi -well plate is fully bonded to the PSA on both the multi -well plate side and the glass substrate side and thatthere are no voids connected to the hole features, as such providing the fiducial markers for autocentering.

[0051] FIG. 2 illustrates a detailed look of the pressure sensitive adhesive (PSA) that includes the fiducial markers. Referring to the example in FIG. 2, the PSA comprises a key notch 210 in a comer for assembly orientation with the multi-well plate and the glass substrate. In some embodiments, the PSA comprises a number of through-cut areas 220 that correspond to the geometry of the sample wells of the multi-well plate. In this example of FIG. 2, the PSA shows 96 sample related through-cut squares with a corner key notch. In some embodiments, the PSA may comprise cut circles at the center of where four adjacent squares meet to serve as fiducial markers see also FIG. 4), as shown in the example illustrated in FIG. 2. In some embodiments, the through-cut areas 220 and fiducial markers 230 may be cut into the PSA by laser cutting. In some embodiments, the through-cut areas 220 and the fiducial markers 230 mayWSGR Docket No. 64100-750.601 be cut into the PSA by die cutting. For example, a CO2laser can be calibrated to specific power, focus, and speed, which can be used to laser-cut the PSA to the design geometry as illustrated in the examples of FIGs. 1-4. In some embodiments, die-cutting may be utilized for generating the hole features (e.g., the fiducial markers) in the PSA. For example, in some embodiments, a rotary die-cutting machine comprising a machined steel or aluminum sharp tool can be rolled on the PSA to die-cut the hole features. In some embodiments, the through -cut areas representing the wells of a multi-well plate can be cut into the PSA following the same laser or die-cut process.

[0052] FIG. 3 shows an example of a bottom view of a multi-well plate comprising the multi-well plate component (FIG. 1A), PSA component (FIG. IB), and the glass substrate component (FIG. 1C) showing a key 310, the sample wells 320, and the fiducial markers 330. In some embodiments, the dimension of a well is from about 3 millimeters (mm) wide to about 8 mm wide. In some embodiments, the dimension of a well is more than or equal to about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, 11 mm, 11.5 mm, or 12 mm wide. In some embodiments, the dimension of a well is less than or equal to about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, 11 mm, 11.5 mm, or 12 mm wide. In some embodiments, the area of a well is approximately 9 mm2to approximately 64 mm2. In some embodiments, the area of a well is more than or equal to 5 mm2, 6 mm2, 7 mm2, 8 mm2, 9 mm2, 10 mm2, 11 mm2, 12 mm2, 13 mm2, 14 mm2, 15 mm2, 16 mm2, 17 mm2, 18 mm2, 19 mm2, 20 mm2, 25 mm2, 30 mm2, 35 mm2, 40 mm2, 45 mm2, 50 mm2, 55 mm2, 60 mm2, 65 mm2, 70 mm2, 75 mm2, 80 mm2, 85 mm2, or 90 mm2. In some embodiments, the area of a well is less than or equal to 5 mm2, 6 mm2, 7 mm2, 8 mm2, 9 mm2, 10 mm2, 11 mm2, 12 mm2, 13 mm2, 14 mm2, 15 mm2, 16 mm2, 17 mm2, 18 mm2, 19 mm2, 20 mm2, 25 mm2, 30 mm2, 35 mm2, 40 mm2, 45 mm2, 50 mm2, 55 mm2, 60 mm2, 65 mm2, 70 mm2, 75 mm2, 80 mm2, 85 mm2, or 90 mm2.However, it is contemplated that the dimension of a well may change depending on the size and number of wells of the multi-well plate being used. FIG. 4 provides exemplary dimensions for the hole features as seen in FIG. 3 (the fiducial markers 330), which the autocentering pipeline navigates for determining the locations of the fiducial markers. In some embodiments, the hole feature 430 being used as a fiducial marker can be approximately 0.5 mm to approximately 3 mm in diameter, however the dimension of a well and the diameter of a hole feature can change depending on the multi-well plate being used. In some embodiments, the hole features can be more than or equal to 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm in diameter. In some embodiments, the hole features canbe less or than equal to 0.5 mm,WSGR Docket No. 64100-750.6011 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm in diameter. In some embodiments, the distance from the fiducial markers to the edges of each of the four well corners can be approximately about 1 .19 mm to about 2.86 mm from the center of the fiducial marker hole feature to the corner edge of a well. In some embodiments, the distance from the fiducial markers to the edges of each of the four well corners may be more than or equal to about 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2. 1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, or 3.4 mm from the center of the fiducial marker hole feature to the corner edge of a well. In some embodiments, the distance from the fiducial markers to the edges of each of the four well corners may be less than or equal to about 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, or 3.4 mm from the center of the fiducial marker hole feature to the corner edge of a well. The example fiducial marker 430 of FIG. 4 shows a fiducial center distance to edge of 1 .44 mm. However, the example of FIG. 4 is for example only and the present disclosure is not so limited.

[0053] FIGs. 5A-5C show examples of various perspectives of an example of an assembled multi-well plate. The fully assembled multi-well plate in this example is illustrated in FIG. 5A. An example of a cutaway of the multi-well plate is shown in FIG. 5B. A side view of a portion of the cutaway of the example multi-well plate is shown in FIG. 5C. In some embodiments, the multi-well plate 510 is superior to the pressure sensitive adhesive (PSA) 520, wherein a fiducial marker 530 is shown in the PSA 520. In some embodiments, the glass substrate 540 may be affixed inferior to the PSA 520. In some embodiments, the multi-well plate 510 top component comprises a skirt 500. In some embodiments, the skirt 500 may be affixed at the bottom of the multi-well plate 510. In some embodiments, the skirt 500 may cover the side of the PSA 520 and the glass substrate 540. As such, in some embodiments, as shown in FIG. 5C, the glass substrate 540 may be recessed up into the multi-well plate component 510 and as such may not be flush (e.g., may be recessed) with the bottom of the multi-well plate skirt 510.

[0054] An example pipeline as described herein for autocentering of an instrument on fiducial markers of the present disclosure is illustrated in FIG. 6. Operations 600, 610, 620, 630, 640, 650, 660, and 670 described herein are non -limiting. As shown in FIG. 6, at operation 600, an image of the fiducials is acquired and converted to grayscale 600. At operation 610, an image quality control check is performed. Quality filters and metrics which may be useful in autocentering may comprise determining the kurtosis or tailedness of the distribution of pixel intensities. The quality filters and metrics may comprise determining the darkness of the image. The quality filters and metrics may comprise determining the radius of the fitted curve. TheWSGR Docket No. 64100-750.601 quality filters and metrics may comprise determining the Intersection over Union (loU) to measure the quality of the circle mask.

[0055] In some embodiments, forthe image quality control check 610, two metrics may be computed, the kurtosis and the full width at half maximum of the grayscale image. Kurtosis of the grayscale image may measure the “tailedness” of the distribution of pixel intensities. Higher kurtosis indicates more outliers, while a lower kurtosis indicates a more symmetrical distribution. Valid ranges of kurtosis can be determined empirically and can be applied to reject low quality images with unexpected intensity distributions. Additionally, in some embodiments, the focus of the image may be estimated by calculating the full width at half maximum (FWHM) of the grayscale image. The median FWHM value, if too high, may indicate autofocus failures and may indicate the image is out of focus and blurry, which may impact the quality of the algorithm. In some embodiments, both the kurtosis and the FWHM values of the image are part of the algorithm output.

[0056] Referring to the example pipeline of FIG. 6, at operation 620, the background is subtracted from the grayscale image. In some embodiments, the background subtraction 620 estimates the background of the input grayscale image using a specified window size. In some embodiments, the estimated background is subtracted from the original image to highlight the foreground features. At operation 620, a check for image brightness may be conducted by determining if the image is too dark based on the intensity percentiles and ratios. In some embodiments, if the image is too dark, a warning may be logged by the instrument software.

[0057] Referring to the example pipeline of FIG. 6, at operation 630, the region of interest (RO I) in the image, that which comprises the fiducial markers, can be cropped by removing the upper and lower regions of the image based on a specified margin size. In some embodiments, the specified margin size is the algorithmic parameter which serves to crop the region where the circle finding is performed, such that the algorithm is able to discern a rounded well corner from a circle to be fitted.

[0058] Referring to the example pipeline of FIG. 6, at operation 640, the foreground of the image is extracted by applying the image to a specified threshold. At operation 650, in some embodiments, a circle fitting process is performed. In some embodiments, after the image has been optimized for further analysis, the fiducial markers, in this example, the fiducial markers are circles, can be fitted for circle fitting. In some embodiments, a random sample consensus (RANSAC) algorithm can be used to fit the circles to the foreground image and update the result, as such outliers of the observed data may be accorded no influence on the results.

[0059] Referring to the example pipeline of FIG. 6, at operation 660, a radius check and loU process is performed. In some embodiments, once the circles are fitted, a radius check and anWSGR Docket No. 64100-750.601 evaluation of the intersection of union (IoU=areaof overlap / area of union) for determining the intersection of the overlap metric is calculated. In some embodiments, the two data are added to the algorithm output.

[0060] Referring to the example pipeline of FIG. 6, at operation 670, the results are collated and reported, and the images can be debugged, if necessary, based on the results for identifying the fiducial markers.

[0061] Example outputs of the circle finding and fitting algorithm illustrated in the example of FIG. 6 are shown in the examples of FIGs. 7A-7D. In some embodiments, the intersection of union, or loU (loU = area of overlap / area of union), is calculated on several fiducial markers of different quality, with the ground truth illustrated in the image of FIG. 7D. In this example, an loU score of <0.1 may be considered a failed circle fit (FIG. 7A), whereas an loU score of >0. 1 (FIG. 7B-7C) may be considered a workable circle fit for the analysis pipeline of FIG. 6. It is contemplated that if an arbitrary object in an image is considered to be a valid circle, the loU score may be low as the arbitrary object is not a valid circle. Similarly, if a valid circle is not auto-fluorescing or, for example, manufacturing defects exist, the loU score may be low. As such, in some embodiments, determining an loU score serves to identify when an object is not a valid circle or when there is some issue associated with a valid circle. As such, in some embodiments, loU scores can provide information for down -weighting of poor registration results and up-weighting of valid registration results, generating a robust workflow for plate registration.

[0062] For applying the example pipeline of FIG. 6 to a multi-well plate comprising a PSA that comprises fiducial markers (e.g., circles located between four adjacent well corners), the fiducial markers analyzed are shown in the example of FIG. 8. Referring to the example of FIG. 8, 18 different fiducial markers located in rows A, B and C and columns 1 -6 were analyzed for intersection of union versus z-offset in four fluorescent channels for a total of 72 images, to determine which channel or channels are best suited for circle finding qualities.

[0063] FIG. 9 shows exemplary results for the four-color channel analysis of the 72 images illustrated in FIG. 8. Referringto FIG. 9, each dotrepresents one of the fiducials as identified in FIG. 8. Each graph of FIG. 9 represents a different fluorescent detection channel (e.g., channel 1, channel 2, channel 3, and channel 4) wherein the x-axis is z-offset and the y-axis is Intersection of Union (loU). In this example, it was determined that channel 1 and channel 2 were the favored choices and that channels 3 and 4 were not as useful for analyzing fiducial markers. In this example, it was determined that channel 3 was the most undesirable channel. Looking further at each fiducial marker from FIG. 8 as identified in channel 1 , FIG. 10 shows examples of 18 graphs corresponding to the rows A, B, and C and columns 1, 2, 3, 4, 5, and 6WSGR Docket No. 64100-750.601(as illustrated in FIG. 8) wherein the x-axis is z-offset and the y-axis represents the Intersection of Union (loU). In this example, FIG. 10 shows that Row B had the overall best performance for circle fitting. In this example, there was no correlation found between FWHM and the z- offset in channel 1. In this example, for channels 1 and 2, analyzing circle fitting in Row B demonstrated that the intersection of union was relatively stable regardless of z- offsets, as shown in FIG. 11. In this example, channels 1 and 2 demonstrated the best performance, making channels 3 and 4 unnecessary for consideration, with the middle row in the plate consistently showing the highest overall performance. Further, it was determined that the algorithmic pipelines for circle fitting and autocentering exhibited stability across varying z-stack offsets, allowing it to perform effectively even if the images were slightly out of focus.

[0064] Circle images and their fitted lines from channel 1 are illustrated in the example of FIG. 12. Challenges included detection of the circle edges and thresholding of what is a circle (arrows designate fitted circles). The challenges in this example are addressed in the example pipeline of FIG. 6. Further examples of fitted circles are shown in the examples of FIGs. 13A- 13B. FIG. 13A shows channel 1 images of a fiducial marker (arrows) rendered to grayscale (left image), demonstrating the decomposition of the original grayscale image into its foreground image (middle image) and its background image (right image). As shown in FIG. 13A, the background image (right image) is subtracted from the grayscale image (left image) and results in the foreground image (middle image). In some embodiments, a thresholding algorithm determines the valid on-pixels which can feed into the circle fitting algorithm, resulting in the example images shown in FIG. 13B with fitted circles (arrows). Additionally, FIG. 14A demonstrates an example of an image with background fluorescence. FIG. 14B demonstrates an example of the image of FIG. 14A with background fluorescence removed. FIG. 14C demonstrates an example of the image of FIG. 14B with a fitted circle applied.

[0065] The example fiducial analysis pipeline is further demonstrated in FIG. 15, which shows images from channel 1 of a fiducial marker with images moving from grayscale and quality control through foreground and background subtraction (as previously described in FIG. 14), followingby application of a random sample consensus (RANSAC) algorithm resulting in a fitted circle to a fiducial marker. The fitted circle pixel intensity distribution is shown in an example graph of FIG. 16 and demonstrates the tight correlation of the frequency vs pixel intensity for the fitted circle of FIG. 15. The graph in FIG. 16 shows the pixel intensity distribution wherein the x-axis represents pixel intensity distribution, and the y-axis represents frequency.WSGR Docket No. 64100-750.601

[0066] An implementation of the autocentering pipeline on a fiducial marker is illustrated in the examples of FIGs. 17A-17B and FIG. 18. Operations 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, and 1790 of FIG. 17A-17B are non-limiting.

[0067] Referring to the example pipeline of FIG. 17A, at operation 1710, an existing good focus model can be uploaded into the imaging instrument. The imaging system can focus on a position as defined by the pipeline at operation 1720, and the imaging can iterate through well positions as shown in FIG. 17B. For example, the imaging can iterate through well positions by moving through the well positions 1 through 6 (illustrated in FIG. 17B).

[0068] Referring to the example pipeline of FIG. 17A, at operation 1730, an autofocusing process is performed. In this example, the imaging system autofocuses on a position, followed by the application of the circle finding algorithm of operation 1740. This is shown in the example of FIG. 6. In this example, as long as three out of the six wells can be measured, and the wells are not collinear, position measurement is considered successful.

[0069] Referring to the example pipeline of FIG. 17A, at operation 1750, after the circle is fitted, the position and confidence of the fiducial marker is recorded by instrument software, which is repeated for each position to be autocentered. After all positions have been autocentered at operation 1760, the expected positions versus the found positions are tabulated, and pixels are converted to millimeters by way of a magnification converter at operation 1770. The data is applied to affine transformation at operation 1780 which can provide insight into any substrate deformations, followed by on instrument storage of the affine transform for potential future use at operation 1790.

[0070] In some embodiments, the affine transform in 2D geometry is a 3x3 matrix that can be computed once three or more pairs of reference and target positions are known. The more data, the more accurate and robust the affine transform 3x3 matrix parameters will be. For example, once the measured positions from the circle fitting algorithm is tabulated, the expected ideal positions of where the circles are generated from the well plate design parameters (e.g., spacing, X, Y location) can be determined. Combining the measured and expected positions of the circles generates pairs of positions that can be input into an affine transform estimation algorithm to estimate the transformation matrix (e.g., translation, rotation, shear, scaling, skew). The affine transform can be stored into a computer software. In the control software, to get the desired well position, matrix multiplication of the affine transform matrix can be performed with the original ideal coordinate space X,Y positions of a circle to arrive at the actual coordinate space locations of a circle, having corrected for translation, rotation, scaling, skew and shear deformations of the substrate.WSGR Docket No. 64100-750.601

[0071] In some embodiments, the image registration and affine transform estimation pipeline illustrated in FIGs. 17A-17B can be used for an autocentering pipeline of the instrument imaging system based on fiducial position, an example of using the determined affine transform for more accurate well position is shown in the example of FIG. 18. Referringto FIG. 18, operations 1810, 1820, and 1830 are non-limiting.

[0072] In some embodiments, the instrument control software loads the pre-computed affine transform model. In some embodiments, to use the model in an x, y, move to some arbitrary well position, the initial well position is first converted into a x, y coordinate location in engineering units (e.g., mm) at operation 1810. In some embodiments, the affine transform model determined from the example pipeline illustrated in FIGs. 17A-17B can be applied to the ideal x, y coordinate locations via matrix multiplication which results in a corrected x, y position (x_c and y_c) at operation 1820. In some embodiments, the instrument control software issues a move command for the imaging system to move to a new well position based on the corrected coordinates x_c and y_c instead of the original ideal locations at operation 1830. As such, the positioning error that may be due to various imperfections of the systems is accounted for via the coordinate transformation, thereby enabling more optimal and accurate imaging and fluid dispensing.

[0073] Various modifications and variations of the disclosed methods, compositions and uses as disclosed herein will be apparent to the skilled person without departing from the scope and spirit of the inventive concepts. Although methods have been disclosed in connection with specific preferred aspects or embodiments, it should be understood that methods, compositions and systems as claimed should not be unduly limited to such specific aspects or embodiments. Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims

Claims

WSGR Docket No. 64100-750.601CLAIMSWhat is Claimed:1 . A multi-well plate configuration comprising:(a) a multi-well plate component comprising a plurality of wells;(b) a substrate component configured to receive biological samples; and(c) an autofluorescent pressure sensitive adhesive component, wherein the autofluorescent pressure sensitive adhesive component comprises a plurality of hole features positionally located between corners of adjacent wells of the plurality of wells.

2. The multi-well plate configuration of claim 1, wherein the hole features of the plurality of hole features serve as fiducial markers.

3. The multi-well plate configuration of claim 1 , wherein the multi -well plate component is affixed superior to the autofluorescent pressure sensitive adhesive component and the substrate component is affixed inferior to the autofluorescent pressure sensitive adhesive component.

4. The multi -well plate configuration of claim 3, wherein the substrate component comprises glass.

5. The multi-well plate configuration of claim 4, wherein the glass is optically clear glass.

6. The multi -well plate configuration of claim 1, wherein the autofluorescent pressure sensitive adhesive component autofluoresces between about 400 nm and about 650 nm.

7. The multi -well plate configuration of claim 1, wherein the autofluorescent pressure sensitive adhesive component comprises a thermoplastic polymer carrier film.

8. The multi-well plate configuration of claim 7, wherein the thermoplastic polymer carrier film comprises one or more of polyester, polypropylene, polyethylene terephthalate, or biaxially oriented polypropylene.

9. The multi-well plate configuration of claim 7, wherein the thermoplastic polymer carrier film comprises an adhesive on a first side of the thermoplastic polymer carrier film and an adhesive on a second side of the thermoplastic polymer carrier film.WSGR Docket No. 64100-750.60110. The multi-well plate configuration of claim 9, wherein the adhesive comprises silicone.11 . The multi-well plate configuration of claim 9, wherein the adhesive comprises acrylate.

12. The multi -well plate configuration of claim 1, wherein the auto fluorescent pressure sensitive adhesive component comprises a thickness of between about 48 pm and about 250 pm.

13. The multi-well plate configuration of claim 1, wherein the hole features of the plurality of hole features comprise a diameter of about 0.5 mm to about 3 mm.

14. The multi -well plate configuration of claim 1, wherein the hole features of the plurality of hole features are laser-cut into the autofluorescent pressure sensitive adhesive component.

15. The multi-well plate configuration of claim 1, wherein the hole features of the plurality of hole features are die-cut into the autofluorescent pressure sensitive adhesive component.

16. The multi -well plate configuration of claim 1, wherein the autofluorescent pressure sensitive adhesive component is transparent.

17. The multi -well plate configuration of claim 1, wherein the autofluorescent pressure sensitive adhesive component is opaque.

18. A method, the method comprising: a) providing a multi-well plate comprising: i) a multi-well component comprising a plurality of wells, wherein the multi -well component is affixed superior to a pressure sensitive adhesive component and ii) a substrate component affixed inferior to the pressure sensitive adhesive component, wherein the pressure sensitive adhesive component comprises a plurality of fiducial markers; b) imaging the multi-well plate, wherein the imaging comprises capturing fluorescent images of the fiducial markers of the plurality of fiducial markers,WSGR Docket No. 64100-750.601 c) registering locations of the fiducial markers of the plurality of fiducial markers of the pressure sensitive adhesive component, and d) determining a location of one or more wells of the plurality of wells for imaging based on the registering of the locations of the fiducial markers.

19. The method of claim 18, further comprising determining a location for fluid dispensing in the one or more wells based at least in part on the registering of the locations of the fiducial markers.

20. The method of claim 18, wherein the plurality of wells comprises between six and 1,536 wells.21 . The method of claim 18, wherein the pressure sensitive adhesive component is double sided.

22. The method of claim 18, wherein the pressure sensitive adhesive component autofluoresces between about 400 nm and about 650 nm.

23. The method of claim 18, wherein the pressure sensitive adhesive component comprises a thermoplastic polymer carrier film.

24. The method of claim 23, wherein the thermoplastic polymer carrier film comprises one or more of polyester, polypropylene, polyethylene terephthalate, or biaxially oriented polypropylene.

25. The method of claim 18, wherein the pressure sensitive adhesive component comprises a thickness of between about 48 pm and about 250 pm.

26. The method of claim 18, wherein the fiducial markers of the plurality of fiducial markers comprise hole features comprising a diameter of approximately about 0.5 mm to about 3 mm.

27. The method of claim 23, wherein the thermoplastic polymer carrier film is transparent.

28. The method of claim 23, wherein the thermoplastic polymer carrier film is opaque.WSGR Docket No. 64100-750.60129. The method of claim 18, further comprising disposing a plurality of biological samples into one or more wells of the plurality of wells.

30. The method of claim 29, wherein the biological samples of the plurality of biological samples comprise nucleic acids, proteins, or a combination thereof.

31. The method of claim 18, wherein the fiducial markers of the plurality of fiducial markers are located equidistant between comers of adjacent wells of the plurality of wells of the multi-well plate component.

32. A composition for centering an imaging device on a well of a multi -well plate, comprising (a) a pressure sensitive adhesive, wherein the pressure sensitive adhesive is autofluorescent, and (b) a plurality of hole features positioned equidistant between comers of adjacent wells of the multi-well plate.

33. A system for registering one or more fiducial markers for a multi -well plate, comprising: a) a computer system comprising instructions for executing a centering program, wherein the centering program is configured to:(i) input a fluorescent image of the one or more fiducial markers from the multi-well plate;(ii) subtract a background from the fluorescent image of the one or more fiducial markers;(iii) determine a region of interest in the fluorescent image of the one or more fiducial markers;(iv) extract a foreground from the region of interest in the fluorescent image of the one or more fiducial markers;(v) circle fit the one or more fiducial markers;(vi) determine a radius of the one or more fiducial markers; and(vii) determine an intersection of union of the one or more fiducial markers; and b) register the one or more fiducial markers based at least in part on (i)-(vii) of the centering program.

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