Methods and compositions for assessing performance of instruments suitable for single molecule tracking

Abstract

The present disclosure relates to a composition comprising a plurality of individually addressable reference samples, the individually addressable reference samples are selected from the group consisting of a reference sample comprising a plurality of spatially stationary optical point sources, a reference sample comprising a plurality of diffusible optical point sources, a reference sample comprising a uniform optical volume, and a reference sample comprising one or more resolution test patterns. The optical point source may be a fluorescent point source, such as a quantum dot or nanodiamond. The uniform optical volume may contain fluorescent molecules. The disclosure also relates to a method of analyzing imaging parameters, acquisition parameters, and / or single molecule tracking (SMT) parameters based on captured images by capturing images of the individually addressable reference sample of the composition with a microscope, and methods of integrating the analyses to assess performance characteristics of the microscope to assess a plurality of performance characteristics of the microscope.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 538,056, dated September 12, 2023, the contents of which are incorporated herein by reference in their entirety, and claims protection of that priority. Technical Field

[0003] The topics described herein relate to methods and compositions for evaluating the performance of instruments suitable for tracking single molecules. Background Technology

[0004] Molecular motion is heavily influenced by its interaction with its surrounding environment. Single-molecule tracking (SMT) is a method for capturing molecular motion as an active reporter. In SMT, target fluorescent molecules are imaged at high spatiotemporal resolution to track their motion. This tracking can occur in relatively simple systems, such as simple buffer solutions, or in complex systems, such as living cells. The information embedded in these tracks has been used to study a variety of molecular properties, including protein-protein interactions, such as interactions mediating signal transduction, inter-organelle communication, nuclear organization, and transcriptional regulation. Given the nature of imaging performed in the context of SMT analysis, accurate performance evaluation of SMT instrument platforms is required. Therefore, there remains a need in the art for effective and efficient methods and compositions for accurately evaluating the performance of such instruments suitable for SMT analysis. Summary of the Invention

[0005] In a first aspect, this disclosure relates to methods and compositions for evaluating the performance of instruments suitable for tracking single molecules.

[0006] In some embodiments, this disclosure relates to compositions for evaluating the performance of instruments suitable for tracking single molecules within complex systems, the compositions comprising a plurality of individually addressable reference samples. In some embodiments, the individually addressable reference samples used in the compositions for evaluating the performance of instruments suitable for tracking single molecules are selected from: reference samples comprising a plurality of spatially stationary optical point sources; reference samples comprising a plurality of diffusible optical point sources; reference samples comprising a uniform optical volume; and reference samples comprising one or more resolution test patterns.

[0007] In some embodiments, the reference sample used in the compositions of this disclosure is housed in an individually addressable sample chamber of a sample container. In some embodiments, each of such sample chambers includes: a bottom surface made of an optically permeable material; and a vertical wall formed along the periphery of the bottom surface, in which a cavity is formed by a closed bottom end and an open top end.

[0008] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of spatially stationary optical point sources immobilized in a polymer. In some embodiments, the polymer in which the plurality of spatially stationary optical point sources are immobilized is a hydrogel. In some embodiments, the polymer in which the plurality of spatially stationary optical point sources are immobilized is a polysaccharide hydrogel, polyacrylamide (PAA) hydrogel, polyacrylic acid hydrogel, polymethyl methacrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or combinations thereof. In some embodiments, the polymer in which the plurality of spatially stationary fluorescent point sources are immobilized is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene, polycarbonate, or polyvinyl butyral. In some embodiments, the polymer in which the plurality of spatially stationary optical point sources are immobilized is degassed or homogenized and then degassed.

[0009] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of spatially stationary optical point sources immobilized in a polymer via covalent bonding, adsorption, electrostatic bonding, or a combination thereof. In some embodiments, the polymer in which the plurality of spatially stationary optical point sources are immobilized comprises a 3D dot matrix.

[0010] In some embodiments of this disclosure, the plurality of spatially stationary optical point sources are multiple spatially stationary fluorescent point sources. In some embodiments of this disclosure, the plurality of spatially stationary fluorescent point sources are composed of quantum dots (Qdots). In some embodiments, the quantum dots have an emission maximum of about 400 nm to about 720 nm. In some embodiments, the quantum dots have an emission maximum of about 420 nm to about 480 nm. In some embodiments, the quantum dots have an emission maximum of 450 nm. In some embodiments, the quantum dots have an emission maximum of about 500 nm to about 550 nm. In some embodiments, the quantum dots have an emission maximum of 525 nm. In some embodiments, the quantum dots have an emission maximum of 545 nm. In some embodiments, the quantum dots have an emission maximum of about 525 nm to about 575 nm. In some embodiments, the quantum dots have an emission maximum of 565 nm. In some embodiments, the quantum dots have an emission maximum of about 575 nm to about 650 nm. In some embodiments, the quantum dots have an emission maximum of 585 nm. In some embodiments, the quantum dots have an emission maximum of 605 nm. In some embodiments, the quantum dot has an emission maximum of 625 nm. In some embodiments, the quantum dot has an emission maximum of about 650 nm to about 720 nm. In some embodiments, the quantum dot has an emission maximum of 655 nm. In some embodiments, the quantum dot has an emission maximum of 705 nm.

[0011] In some embodiments, multiple spatially stationary fluorescent spot sources disposed in multiple individually addressable reference samples are composed of nanodiamonds. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 400 nm to about 720 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 400 nm to about 500 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 415 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 500 nm to about 550 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 550 nm to about 600 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of 510 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of 575 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 600 nm to about 700 nm. In some embodiments, the nanodiamond spatially stationary fluorescent dot source has an emission maximum of 638 nm.

[0012] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of diffusing optical spot sources disposed within a solution. In some embodiments, the solution in which the diffusing optical spot sources are disposed comprises Tris:HCl, DMSO, DMEM, DPBS, or H2O. In some embodiments, the H2O is distilled H2O or deionized H2O. In some embodiments, the solution in which the diffusing optical spot sources are disposed comprises polytungstate. In some embodiments, the solution in which the diffusing optical spot sources are disposed comprises TWEEN® 20 (polysorbate 20). In some embodiments, the solution in which the diffusing optical spot sources are disposed comprises Triton X-100® (2-[4-(2,4,4-trimethylpentane-2-yl)phenoxy]ethanol).

[0013] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of diffusing optical point sources, wherein each diffusing optical point source contains a conjugated compound. In some embodiments, the compound conjugated with the diffusing optical point source is a protein, polyethylene glycol, polysaccharide, oligonucleotide, polyamine, or polyamino acid, or derivatives thereof and analogs thereof.

[0014] In some embodiments, the compound conjugated with the diffusible optical point source is a protein selected from ovalbumin, serum albumin, and avidin.

[0015] In some embodiments, the compound conjugated with the diffusing optical point source is a polyamino acid selected from polylysine, polyhistidine, or polyglutamic acid, polyaspartic acid, their derivatives, and their analogues. In some embodiments, the compound conjugated with the diffusing optical point source is polyethylene glycol, its derivatives, and their analogues with a molecular weight of about 200 Da to about 6000 Da.

[0016] In some embodiments of this disclosure, the reference sample comprises a plurality of diffusing optical point sources, each of which comprises a conjugated compound. The reference sample is housed in an individually addressable chamber, and the compound conjugated to the diffusing optical point source is operatively connected to a chamber surface in contact with the reference sample. In some embodiments of this disclosure, the reference sample comprises a plurality of diffusing optical point sources, each of which comprises a conjugated compound. The reference sample is housed in an individually addressable chamber, and the compound is conjugated to the chamber surface and to a diffusing fluorescent point source. In some embodiments of this disclosure, the reference sample comprises a plurality of diffusing optical point sources, each of which comprises a conjugated compound. The compound is conjugated to the chamber surface and to a diffusing optical point source, with a first end conjugated to the bottom surface of the sample chamber and a second end conjugated to the diffusing optical point source. In some embodiments, conjugation is performed by covalent bonding, adsorption, electrostatic bonding, hydrophobic bonding, or a combination thereof.

[0017] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of diffusing optical point sources disposed within a polymer. In some embodiments, the polymer in which the plurality of diffusing optical point sources are disposed is a hydrogel. In some embodiments, the hydrogel in which the plurality of diffusing optical point sources are disposed is a polysaccharide hydrogel, polyacrylamide hydrogel, polyacrylic acid hydrogel, polymethyl acrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or combinations thereof. In some embodiments, the hydrogel in which the plurality of diffusing optical point sources are disposed is a polyacrylamide hydrogel or an agarose hydrogel. In some embodiments, the hydrogel in which the plurality of diffusing optical point sources are disposed is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene (PTFE), polycarbonate, or polyvinyl butyral.

[0018] In some embodiments of this disclosure, the plurality of diffusing optical dot sources are plurality of diffusing fluorescent dot sources. In some embodiments of this disclosure, the plurality of diffusing fluorescent dot sources comprise quantum dots. In some embodiments, the quantum dots have an emission maximum of about 400 nm to about 720 nm. In some embodiments, the quantum dots have an emission maximum of about 420 nm to about 480 nm. In some embodiments, the quantum dots have an emission maximum of 450 nm. In some embodiments, the quantum dots have an emission maximum of about 500 nm to about 550 nm. In some embodiments, the quantum dots have an emission maximum of 525 nm. In some embodiments, the quantum dots have an emission maximum of 545 nm. In some embodiments, the quantum dots have an emission maximum of about 525 nm to about 575 nm. In some embodiments, the quantum dots have an emission maximum of 565 nm. In some embodiments, the quantum dots have an emission maximum of about 575 nm to about 650 nm. In some embodiments, the quantum dots have an emission maximum of 585 nm. In some embodiments, the quantum dots have an emission maximum of 605 nm. In some embodiments, the quantum dot has an emission maximum of 625 nm. In some embodiments, the quantum dot has an emission maximum of about 650 nm to about 720 nm. In some embodiments, the quantum dot has an emission maximum of 655 nm. In some embodiments, the quantum dot has an emission maximum of 705 nm.

[0019] In some embodiments, the multiple diffusible fluorescent dot sources comprise nanodiamonds. In some embodiments, the nanodiamonds have an emission maximum of about 400 nm to about 720 nm. In some embodiments, the nanodiamonds have an emission maximum of about 400 nm to about 500 nm. In some embodiments, the nanodiamonds have an emission maximum of about 415 nm. In some embodiments, the nanodiamonds have an emission maximum of about 500 nm to about 550 nm. In some embodiments, the nanodiamonds have an emission maximum of 510 nm. In some embodiments, the nanodiamonds have an emission maximum of about 550 nm to about 600 nm. In some embodiments, the nanodiamonds have an emission maximum of 575 nm. In some embodiments, the nanodiamonds have an emission maximum of about 600 nm to about 700 nm. In some embodiments, the nanodiamonds have an emission maximum of 638 nm.

[0020] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a uniform optical volume disposed within a matrix. In some embodiments, the matrix in which the uniform optical volume is disposed is a solution or a polymer. In some embodiments, the matrix in which the uniform optical volume is disposed is a solution comprising Tris:HCl, DMSO, DMEM, DPBS, or H2O. In some embodiments, the H2O is distilled H2O or deionized H2O. In some embodiments, the polymer in which the uniform optical volume is disposed is a sol-gel, gel, or solid. In some embodiments, the polymer matrix in which the uniform optical volume is disposed is a hydrogel. In some embodiments, the hydrogel in which the uniform optical volume is disposed is a polysaccharide hydrogel, polyacrylamide hydrogel, polyacrylic acid hydrogel, polymethyl methacrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or combinations thereof. In some embodiments, the polymer matrix in which the uniform optical volume is disposed is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene (PTFE), polycarbonate, or polyvinyl butyral. In some embodiments, the hydrogel in which the uniform optical volume is disposed is a polyacrylamide hydrogel or an agarose hydrogel. In some embodiments, the hydrogel in which the uniform optical volume is disposed is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene (PTFE), polycarbonate, or polyvinyl butyral.

[0021] In some embodiments, the uniform optical volume is a uniform fluorescence volume. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 400 nm to about 720 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 420 nm to about 480 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of 450 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 500 nm to about 550 nm. For example, in a particular embodiment, the fluorescent molecule is a fluorescein having an emission maximum of 517 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 525 nm to about 575 nm. For example, in a particular embodiment, the fluorescent molecule is rhodamine B having an emission maximum of 550 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of 565 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 575 nm to about 650 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 650 nm to about 720 nm. In some embodiments, the fluorescent molecules are quenched fluorescent molecules.

[0022] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing one or more resolution test patterns suitable for evaluating one or more of a resolution test pattern, a field distortion test pattern, or a parfocal stability test pattern. For example, but not as a limitation, the one or more resolution test patterns may be selected from absorptive patterns, reflective patterns, and birefringent patterns. Exemplary resolution test patterns known in the art include, but are not limited to, the following: USAF 1951 resolution test pattern; NBS 1952 resolution test pattern; Ronchi scribed target; and fan-shaped star target.

[0023] In some embodiments, this disclosure relates to a method for evaluating multiple performance characteristics of an instrument suitable for tracking single molecules using a composition comprising multiple individually addressable reference samples. In some embodiments, the individually addressable reference samples used in the composition for evaluating the performance of an instrument suitable for tracking single molecules are selected from: reference samples comprising multiple spatially stationary optical point sources; reference samples comprising multiple diffusible optical point sources; reference samples comprising a uniform optical volume; and reference samples comprising one or more resolution test patterns. In some embodiments of such methods, the instrument is a microscope. In some embodiments, such methods include: aligning a microscope objective with one of the multiple individually addressable reference samples, wherein the vertical axis of the objective is perpendicular to the horizontal axis of the sample; and analyzing one or more imaging parameters based on images captured by the microscope; analyzing one or more acquisition parameters based on images captured by the microscope; analyzing one or more SMT analysis parameters based on images captured by the microscope; or a combination thereof; repeating the alignment and analysis steps for the multiple individually addressable reference samples; and integrating the analyses performed on the multiple individually addressable reference samples, thereby evaluating multiple microscope performance characteristics.

[0024] In some embodiments, the methods of this disclosure involve evaluating multiple performance characteristics of an instrument suitable for tracking single molecules, wherein the method includes analyzing one or more imaging parameters. In some embodiments, imaging parameters include: laser parameters, alignment parameters, camera parameters, detection parameters, or combinations thereof. In some embodiments, analyzing laser parameters includes analyzing one or more of laser wavelength, laser pulse, and laser pulse duration. In some embodiments, analyzing alignment parameters includes analyzing one or both of sheet characteristics and focal plane. In some embodiments, analyzing sheet characteristics includes analyzing one or more of: sheet thickness; sheet uniformity; sheet tilt angle; and sheet intensity density. In some embodiments, analyzing camera parameters includes analyzing one or more of camera noise mode, relative alignment, orientation, or magnification. In some embodiments, analyzing detection parameters includes analyzing one or more of aberrations, sensitivity, and resolution. In some embodiments, analyzing aberrations includes analyzing one or more of spherical aberration, chromatic aberration, coma aberration, and cloverleaf aberration. In some embodiments, analyzing the focal plane includes analyzing the position, flatness, orientation relative to the sample and detector, and thickness of the optical focal plane.

[0025] In some embodiments, the methods of this disclosure involve evaluating multiple performance characteristics suitable for tracking single molecules, wherein the method includes analyzing one or more acquisition parameters of the instrument. In some embodiments, the acquisition parameters of the analytical instrument include analysis time parameters and / or spatial parameters. In some embodiments, the analysis time parameters include one or more of analysis frame rate, exposure time, number of frames, or channels. In some embodiments, the analysis spatial parameters include one or more of the following: analysis field of view (FOV) size on the detector chip, FOV position and orientation on the detector chip, FOV position or FOV orientation in the sample.

[0026] In some embodiments, the methods of this disclosure involve evaluating multiple performance characteristics suitable for tracking single molecules, wherein the method includes analyzing one or more SMT parameters of the instrument. In some embodiments, analyzing SMT parameters includes analyzing single-molecule localization and / or single-molecule tracking. In some embodiments, analyzing single-molecule localization includes analyzing one or more of localization error, spot count, and signal-to-noise ratio (SNR). In some embodiments, analyzing single-molecule tracking includes analyzing one or more of trajectory count, trajectory length, jump length, and mean posterior diffusion coefficient.

[0027] In some embodiments, the methods of this disclosure involve evaluating multiple performance characteristics suitable for instruments tracking single molecules, wherein the performance characteristics are selected from: tilt line scan (OLS) alignment characteristics: rotation relative to the camera, position in the FOV, tilt angle, excitation flux; tilt line scan (OLS) uniformity characteristics: coefficient of variation (CV), sag (the vertical height difference between the support point of the line (e.g., the laser line at the edge of the field of view) and the lowest point), fringes, thickness, mechanical and temperature effects; detection characteristics: background and camera noise, point spread function (PSF), signal-to-noise ratio (SNR), diffusion characteristics, optical aberrations, correction ring settings, spatial resolution, Strell ratio, FOV uniformity, camera rotation relative to the sample, position and orientation of the focal plane relative to the objective and detector, focal plane thickness and flatness; acquisition characteristics: plate position and levelness, scan amplitude, scan offset, galvo scanning, camera synchronization. In some embodiments, the instrument’s performance (imaging parameters, acquisition parameters, and single-molecule tracking (SMT) parameters) is compared with reference parameter values, where deviations from the reference parameter values ​​indicate the need to adjust the microscope, and the method further includes automatic adjustment of the microscope based on individual integrated analysis or based on comparison with a reference. Attached Figure Description

[0028] Figure 1 The compositions disclosed herein are shown, comprising: (1) a diffusible fluorescent spot source; (2) a spatially stationary fluorescent spot source; or (3) a controlled dynamic fluorescent spot source.

[0029] Figure 2 The present disclosure illustrates a composition comprising a plurality of individually addressable reference samples present at a specific location (e.g., at a specific location in a porous microplate). Figure 2 Exemplary performance characteristics that can be analyzed in conjunction with the compositions of this disclosure are also indicated. The specific location for analyzing any particular performance characteristic is not fixed and is alternatively arranged within the scope of this disclosure.

[0030] Figure 3 A schematic diagram depicts an SMT workflow according to a specific embodiment of the present disclosure.

[0031] Figures 4A to 4D A schematic diagram of an exemplary image acquisition system according to a particular embodiment of the present disclosure is depicted, wherein the XZ plane is visible ( Figure 4A and Figure 4D () or visible in the YZ plane () Figure 4B and Figure 4C ).

[0032] Figures 5A to 5E An illustration depicts an example optical engine component of this disclosure according to a particular embodiment.

[0033] Figures 6A to 6B Various metrics are described to demonstrate that the image acquisition system and workflow of this disclosure are suitable for robust high-throughput single-molecule tracking (htSMT) analysis. Figure 6A The cross-sectional characteristics of an example beam for OLS and alternative methods according to a specific embodiment are shown. Figure 6B The rolling shutter operation according to a particular embodiment is illustrated, which depicts an example of the relative timing and synchronization of sensor exposure and sample illumination scanning.

[0034] Figures 7A to 7E Representative results from the alignment parameter analysis are presented. Figure 7A Alignment analysis for an example tilt angle (AOI) of the light sheet is described. Figure 7B Alignment analysis for example galvanometer scanning synchronization is described. Figure 7C The alignment procedure for galvanometer scanning synchronization in an iterative example is described. Figure 7D Alignment analysis is depicted for example spatial distributions of SNR and PSF. Figure 7E The distribution and statistics of example optical sheets according to a specific embodiment of this disclosure are depicted.

[0035] Figure 8 A schematic diagram of an exemplary sample processing system of this disclosure is depicted.

[0036] Figure 9 An exemplary system for a high-throughput single-molecule imaging platform for measuring protein motion in living cells is shown.

[0037] Figure 10 The data stream of an exemplary system is shown through a high-throughput single-molecule imaging platform used to measure protein motion in living cells.

[0038] Figure 11 Several images depict the differences between mask categories and instance or semantic masks.

[0039] Figure 12 An example computer implementation environment relevant to the topics described herein is shown.

[0040] Figure 13 This is a diagram illustrating the architecture of a sample computing device used to implement the various aspects described herein.

[0041] It should be noted that the accompanying drawings are schematic rather than literal or precise; the components and aspects of the drawings may not necessarily be drawn to scale. Furthermore, while the same reference numerals may indicate corresponding parts in different views in many cases, the same parts may not always be provided with the same reference numerals in every view. Detailed Implementation

[0042] The currently disclosed subject matter relates to methods and compositions for evaluating the performance of instruments suitable for tracking single molecules. For example, but not as a limitation, this disclosure relates to compositions for evaluating the performance of instruments suitable for tracking single molecules within complex systems, the compositions comprising a plurality of individually addressable reference samples, and methods for using such compositions. In some embodiments, the individually addressable reference samples used in the compositions for evaluating the performance of instruments suitable for tracking single molecules are selected from: reference samples comprising a plurality of spatially stationary optical (e.g., fluorescent) point sources; reference samples comprising a plurality of diffusible optical (e.g., fluorescent) point sources; reference samples comprising a uniform optical (e.g., fluorescent) volume; and reference samples comprising a plurality of absorptive or reflective patterns. In some embodiments, the compositions and methods of this disclosure will employ a plurality of reference samples comprising one or more of the following: reference samples comprising a plurality of spatially stationary optical (e.g., fluorescent) point sources; reference samples comprising a plurality of diffusible optical (e.g., fluorescent) point sources; reference samples comprising a uniform optical (e.g., fluorescent) volume; and reference samples comprising a plurality of absorptive or reflective patterns.

[0043] The subject matter of this disclosure is described with reference to the accompanying drawings, wherein reference numerals are consistently used to indicate similar or equivalent elements. The drawings are not drawn to scale and are provided solely to illustrate the aspects disclosed herein. Several aspects of the disclosure are described below with reference to exemplary hardware, software, and applications for illustrative purposes. It should be understood that numerous specific details, relationships, and methods are set forth to provide a more complete understanding of the subject matter disclosed herein. For clarity and not limitation, the detailed description is divided into the following subsections:

[0044] 1. Definition

[0045] 2. Evaluate the performance of instruments suitable for single-molecule tracking.

[0046] 3. Exemplary single-molecule tracking analysis hardware

[0047] 4. Exemplary Single-Molecular Tracking Analysis Software

[0048] 5. Example

[0049] 1. Definition

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, this document (including the definitions) shall prevail. While preferred methods and materials are described below, similar or equivalent methods and materials may be used in the implementation or testing of the subject matter disclosed herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0051] As used herein, the terms “comprising,” “including,” “having,” “having,” “may,” “containing,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. This disclosure also contemplates other instances of “comprising” the examples or elements presented herein, “consisting of the examples or elements presented herein,” and “consisting substantially of the examples or elements presented herein,” whether or not explicitly stated.

[0052] In describing the numerical ranges in this paper, each intermediate number within the range is explicitly considered with the same degree of precision. For example, for the range of 6-9, the numbers 7 and 8 are considered in addition to 6 and 9; and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.

[0053] As used herein, the terms "about" or "approximately" mean within an acceptable margin of error for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, according to practice in this art, "about" may mean within 3 or greater than 3 standard deviations. Alternatively, "about" may mean a range up to 20% of a given value, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1%. Alternatively, particularly for biological systems or processes, the term may mean within orders of magnitude of the value, preferably within 5 times and more preferably within 2 times.

[0054] As defined herein, molecular “motion” refers to a change in the position and / or orientation of a molecule. In some embodiments, molecular motion can be quantified by analyzing changes in spatial coordinates over consecutive time points (e.g., trajectory analysis). Motion characterized in this way can also include, but is not limited to, measurements of diffusion coefficients. For example, but not as a limitation, the measurement can be a maximum likelihood estimate of the diffusion coefficient, defined as an estimate of the maximum likelihood diffusion coefficient for multiple trajectories under a singlet diffusion model with constant localization error. In some embodiments, molecular motion can be measured by analyzing the product of link generation algorithms. Motion characterized in this way can include, but is not limited to, the mean posterior diffusion coefficient, or the average of the posterior probability distributions of coefficients from a probabilistic linking algorithm. Motion characterized in this way can include, but is not limited to, the geometric mean posterior diffusion coefficient, or the average of the log-scaled posterior probability distributions of coefficients from a probabilistic linking algorithm. Motion characterized in this way can include, but is not limited to, measurements of jump length distributions. For example, for a given set of protein displacements between one time point and subsequent time points, a histogram of the probability of each of the displacement lengths (“jump lengths”) can be constructed. The quantiles of this distribution can be used to describe molecular motion. In some embodiments, the quantile used is the median of the jump length distribution. In some embodiments, the quantile used is the third quartile of the jump length distribution. Motion characterized in this way may include, but is not limited to, measurements of mean square displacement, defined as the average of the squares of all displacements in the trajectory, taken as the average across multiple trajectories. Motion characterized in this way may also include, but is not limited to, measurements of trajectory length or trajectory length distribution. Motion characterized in this way may also include, but is not limited to, measurements of the mean radius of gyration, defined as the root mean square distance of all coordinates in the trajectory from the centroid of the set of points contained in the trajectory, taken as the average across multiple trajectories. Motion characterized in this way may also include, but is not limited to, measurements of the mean bond angle, defined as the average of the angles formed by three consecutive spatial coordinates across multiple trajectories. In some embodiments, molecular motion can be measured by model dependency analysis of multiple trajectories. Motion characterized in this way may include, but is not limited to, the fraction of immobile molecules ("f") defined by a two-state model fitting. 结合 (”).

[0055] As used herein, the term "motion" includes changes in direction and changes in the target's velocity (both increasing and decreasing). Therefore, in some embodiments, tracking motion may include determining that the target is not moving, for example, when the target is in or substantially in a static binding state. As described above, such motion can be characterized in a variety of ways, including, but not limited to, quantifying (a) the diffusion coefficients of multiple trajectories obtained from maximum likelihood estimators; (b) the geometrically mean posterior diffusion coefficients of multiple trajectories; (c) the median of the jump length distributions of multiple trajectories; (d) the third quartile of the jump length distributions of multiple trajectories; (e) the median radius of rotation of multiple trajectories; (f) the mean posterior diffusion coefficients of multiple trajectories; (g) the mean square displacements of multiple trajectories; (h) the median bond angles of multiple trajectories; or (i) the trajectory lengths of multiple trajectories.

[0056] As used herein, the term "trajectory" refers to the set of spatial coordinates of a molecular observation location corresponding to a linking time. In some embodiments, multiple trajectories can be algorithmically constructed by linking multiple molecules whose positions at consecutive time points are known. In some embodiments, multiple trajectories can be conservatively constructed by linking only points within a fixed search radius when no other reasonable links are available. In some embodiments, multiple trajectories can be constructed probabilistically.

[0057] In some embodiments, molecular motion can be quantified by analyzing changes in the rotational motion of molecules at consecutive time points. For example, but not limited to, motion characterized in this way can be determined by measuring fluorescence polarization. Fluorescence polarization is a measurement of the orientation change of a target or test molecule over the time interval between absorption and emission events. For example, but not limited to, if a fluorophore is excited with polarized light, a slowly rotating molecule containing that fluorophore will emit more light retaining its original polarization than a rapidly rotating molecule. Therefore, by measuring fluorescence polarization, the rotational motion of the target or test molecule can be determined, and this information can be used to calculate the measurement result of the rotational motion.

[0058] In some embodiments, the measurement of the rotational motion described herein is calculated as a function of the anisotropic decay time. The system described herein allows the detection of the anisotropic decay time and can calculate it using strategies known in the art, such as those outlined in "Time-Dependent Anisotropy Decays" (Lakowicz, JR, ed.) Principles of Fluorescence Spectroscopy. Springer, Boston, MA. https: / / doi.org / 10.1007 / 978-0-387-46312-4_11, which are hereby incorporated herein by reference in their entirety. In some embodiments, the measurement of the rotational motion described herein is calculated as a function of the fluorescence polarization intensity change. The system described herein allows the detection of fluorescence polarization intensity changes, which can then be calculated using strategies known in the art. In some embodiments, the measurement of the rotational motion described herein is calculated as a function of the rotational diffusion relaxation time. The system described herein allows the detection of the rotational diffusion relaxation time intensity changes, which can then be calculated using strategies known in the art.

[0059] As used herein, the motion being detected, including but not limited to any change in motion, can occur in response to any environmental or other factor (e.g., the presence of the test molecule). For example, but not as a limitation, motion or lack thereof can be triggered by: (A) the addition of a molecule; (B) a change in temperature; (C) a change in oxygen concentration, e.g., the introduction of hypoxic conditions; (D) mechanical stress; (E) a change in pH; (F) a change in exposure (e.g., an increase or decrease in intensity); and / or (G) a change in solution composition.

[0060] As used herein, the term "multiple" refers to a number greater than one. In some embodiments, the term "multiple target molecules" refers to a number greater than one target molecule. For example, but not by limitation, "multiple target molecules" may include at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 500,000, or at least about 1,000,000 target molecules. In some embodiments, the term "multiple test molecules" refers to a number greater than one test molecule. For example, but not as a limitation, multiple test molecules may include at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 500,000, or at least about 1,000,000 test molecules.

[0061] As used herein, the term "fluorescent protein" refers to any protein that emits a fluorescent signal. In some cases, fluorescence emission occurs in response to exposure to light of a specific wavelength. An example of a naturally occurring fluorescent protein is green fluorescent protein (GFP). However, in some instances, a target protein can be adapted to emit a fluorescent signal by introducing an encoded fluorescent tag, i.e., by fusing a protein sequence to the target protein to make it fluoresce. In some instances, a target protein can be adapted to emit a fluorescent signal by binding a fluorescent ligand. Non-limiting examples of such encoded fluorescent tags include: Halo tags, SNAP tags, CLIP tags, TMP tags, and Sun tags. Alternatively or additionally, a protein can be adapted to emit a fluorescent signal by coupling the target protein to a fluorescent dye molecule (e.g., an amine-reactive or thiol-reactive dye).

[0062] As used herein, the term "compound" refers to any chemically defined entity. In some instances, a compound may be a molecule less than 1000 Da, i.e., a "small molecule". In some instances, a compound may be a macromolecule, such as a nucleic acid. In some instances, a nucleic acid may have a defined sequence. In some instances, nucleic acids include: (A) ribonucleic acid (RNA), including, for example, modified RNA; (B) deoxyribonucleic acid (DNA), including, for example, modified DNA; and (C) a combination of (A) and (B). In some instances, the nucleic acid will be a single-stranded or double-stranded small interfering nucleic acid (e.g., double-stranded siRNA), antisense oligonucleotides, ribozymes, microRNAs, or aptamers. In some instances, a compound may be a protein. For example, but not as a limitation, the protein compounds disclosed herein encompass signaling proteins such as protein hormones, cytokines, kinases, phosphatases, and other enzymes, as well as transcription factors, and antibodies, contractile proteins, structural proteins, storage proteins, and transport proteins. In some instances, a compound may refer to a mixture of molecules, e.g., a mixture with a defined composition.

[0063] As used herein, the term “uniform intensity” refers to light intensity (e.g., light directed toward the sample plane) where the intensity difference is no more than 5% in some instances, no more than 10% in some instances, or no more than 15% in some instances.

[0064] As used in this article, the term “uniform intensity” in relation to signal-to-noise ratio (SNR) refers to the pixel-by-pixel SNR within the field of view (FOV), where possible values ​​range from 0.5 to 1 standard deviation from the average SNR.

[0065] 2. Evaluate the performance of instruments suitable for single-molecule tracking.

[0066] 2.1 Reference Sample Composition

[0067] In some embodiments, this disclosure relates to compositions for evaluating the performance of instruments suitable for tracking single molecules within complex systems, the compositions comprising a plurality of individually addressable reference samples. In some embodiments, the individually addressable reference samples used in the compositions for evaluating the performance of instruments suitable for tracking single molecules are selected from: reference samples comprising a plurality of spatially stationary optical (e.g., fluorescent) point sources; reference samples comprising a plurality of diffusible optical (e.g., fluorescent) point sources; reference samples comprising a uniform optical (e.g., fluorescent) volume; and reference samples comprising a plurality of absorptive or reflective patterns.

[0068] In some embodiments, the reference sample used in the compositions of this disclosure is housed in an individually addressable sample chamber of a sample container. In some embodiments, each of such sample chambers includes: a bottom surface made of an optically permeable material; and a vertical wall formed along the periphery of the bottom surface, in which a cavity is formed by a closed bottom end and an open top end.

[0069] refer to Figure 2 Aspects of the present topic can be implemented using plates, such as 384-well glass plates for tissue culture processing, although other plate types can also be used with the methods outlined herein, including but not limited to single-chamber, 6-well, 8-well, 9-well, 24-well, 96-well, 1536-well, and 3456-well glass plates, as well as plates made of alternative materials, such as plates made partially or entirely of plastic. In some embodiments, such plates will mate with corresponding caps. In some embodiments, the caps will be secured to the plate to prevent loss of contents contained in one or more wells.

[0070] In some embodiments, the sample container (e.g., plate) of this disclosure may be surface-treated. In some embodiments, the sample container may include one or more surface-treated sample chambers and one or more untreated chambers. Exemplary surface treatments used with the sample container of this disclosure include, but are not limited to: ion beam treatment, plasma treatment; UV / ozone treatment; and surface adsorption on polymer substrates (e.g., polyethylene glycol (PEG), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polystyrene (PS), polyvinylidene fluoride (PVDF), etc.). Exemplary surface treatments within the scope of this disclosure include those disclosed in Auld et al., Microplate Selection and Recommended Practices in High-throughput Screening and Quantitative Biology, (2020) in: Assay Guidance Manual edited by Markossian S, Grossman A, Arkin M et al., which are hereby incorporated herein by reference in their entirety. Such surface treatments may be used in some embodiments to prepare (e.g., clean or etch) surfaces for use, while in other embodiments, such surface treatments are used to modify (e.g., attach chemical functional groups and / or biomolecules) surfaces for use.

[0071] 2.1.1. Reference sample containing a spatially stationary optical point source

[0072] In some embodiments, and as Figure 1As shown, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of spatially stationary optical (e.g., fluorescent) point sources fixed in a polymer. In some embodiments, the polymer in which the plurality of spatially stationary optical (e.g., fluorescent) point sources are fixed is a hydrogel. In some embodiments, the polymer in which the plurality of spatially stationary optical (e.g., fluorescent) point sources are fixed is a polysaccharide hydrogel, polyacrylamide hydrogel, polyacrylic acid hydrogel, polymethyl methacrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or combinations thereof. In some embodiments, the polymer in which the plurality of spatially stationary optical (e.g., fluorescent) point sources are fixed is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene, polycarbonate, or polyvinyl butyral. In some embodiments, the polymer in which multiple spatially stationary optical (e.g., fluorescent) point sources are fixed is degassed, or homogenized and degassed.

[0073] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of spatially stationary optical (e.g., fluorescent) point sources, wherein the optical (e.g., fluorescent) point sources are immobilized in a polymer by covalent coupling, adsorption, electrostatic bonding, or a combination thereof. In some embodiments, the polymer in which the plurality of spatially stationary optical (e.g., fluorescent) point sources are immobilized comprises a 3D dot matrix.

[0074] In some embodiments of this disclosure, a plurality of spatially stationary fluorescent dot sources comprise quantum dots. Exemplary quantum dots known in the art and commercially available exhibit emission maximum values ​​suitable for the subject matter disclosed herein. In some embodiments, the quantum dots have emission maximum values ​​of about 400 nm to about 720 nm. In some embodiments, the quantum dots have emission maximum values ​​of about 420 nm to about 480 nm. In some embodiments, the quantum dots have emission maximum values ​​of 450 nm. In some embodiments, the quantum dots have emission maximum values ​​of about 500 nm to about 550 nm. In some embodiments, the quantum dots have emission maximum values ​​of 525 nm. In some embodiments, the quantum dots have emission maximum values ​​of 545 nm. In some embodiments, the quantum dots have emission maximum values ​​of about 525 nm to about 575 nm. In some embodiments, the quantum dots have emission maximum values ​​of 565 nm. In some embodiments, the quantum dots have emission maximum values ​​of about 575 nm to about 650 nm. In some embodiments, the quantum dots have emission maximum values ​​of 585 nm. In some embodiments, the quantum dots have emission maximum values ​​of 605 nm. In some embodiments, the quantum dot has an emission maximum of 625 nm. In some embodiments, the quantum dot has an emission maximum of about 650 nm to about 720 nm. In some embodiments, the quantum dot has an emission maximum of 655 nm. In some embodiments, the quantum dot has an emission maximum of 705 nm.

[0075] In some embodiments, the plurality of spatially stationary fluorescent spot sources comprise nanodiamonds. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 400 nm to about 720 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 400 nm to about 500 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 415 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 500 nm to about 550 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 550 nm to about 600 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of 510 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of 575 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of about 600 nm to about 700 nm. In some embodiments, the nanodiamond spatially stationary fluorescent spot sources have an emission maximum of 638 nm.

[0076] 2.1.2. Reference sample containing a diffusible optical point source

[0077] In some embodiments, and as Figure 1 As shown, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of diffusing optical (e.g., fluorescent) spot sources disposed within a solution. In some embodiments, the solution in which the diffusing optical (e.g., fluorescent) spot sources are disposed comprises Tris:HCl, DMSO, DMEM, DPBS, or H2O. In some embodiments, the H2O is distilled H2O or deionized H2O. In some embodiments, the solution in which the diffusing optical (e.g., fluorescent) spot sources are disposed comprises polytungstate. In some embodiments, the solution in which the diffusing optical spot sources are disposed comprises TWEEN® 20 (polysorbate 20). In some embodiments, the solution in which the diffusing optical spot sources are disposed comprises Triton X-100® (2-[4-(2,4,4-trimethylpentane-2-yl)phenoxy]ethanol). In some embodiments, the solution in which the diffusing optical (e.g., fluorescent) spot sources are disposed comprises polytungstate.

[0078] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of diffusing optical (e.g., fluorescent) point sources, wherein each diffusing optical (e.g., fluorescent) point source comprises a conjugated compound. In some embodiments, such conjugation produces controlled, dynamically diffusing optical (e.g., fluorescent) point sources. For example, but not as a limitation, the extent to which such controlled, dynamically diffusing optical (e.g., fluorescent) point sources can diffuse is a function of the conjugation properties. In some embodiments, the compound conjugated with the diffusing optical (e.g., fluorescent) point source is a protein, polyethylene glycol, polysaccharide, oligonucleotide, polyamine, or polyamino acid, or derivatives thereof and their analogues.

[0079] In some embodiments, the compound conjugated with a diffusible optical (e.g., fluorescent) point source is a protein selected from ovalbumin, serum albumin, and avidin.

[0080] In some embodiments, the compound conjugated with the diffusible optical (e.g., fluorescent) point source is a polyamino acid selected from polylysine, polyhistidine, or polyglutamic acid, polyaspartic acid, their derivatives, and their analogs. In some embodiments, the compound conjugated with the diffusible optical (e.g., fluorescent) point source is polyethylene glycol, its derivatives, and their analogs with a molecular weight of about 200 Da to about 6000 Da.

[0081] In some embodiments of this disclosure, the reference sample comprises a plurality of diffusing optical (e.g., fluorescent) point sources, each of which comprises a conjugated compound. The reference sample is housed in an individually addressable chamber, and the compound conjugated to the diffusing optical (e.g., fluorescent) point source is operatively attached to a chamber surface in contact with the reference sample. In some embodiments of this disclosure, the reference sample comprises a plurality of diffusing optical (e.g., fluorescent) point sources, each of which comprises a conjugated compound. The reference sample is housed in an individually addressable chamber, and the compound is conjugated to both the chamber surface and the diffusing optical (e.g., fluorescent) point source. In some embodiments of this disclosure, the reference sample comprises a plurality of diffusing optical (e.g., fluorescent) point sources, each comprising a conjugated compound conjugated to a chamber surface and to a diffusing optical (e.g., fluorescent) point source, the compound being conjugated at a first end to the bottom surface of the sample chamber and at a second end to the diffusing optical (e.g., fluorescent) point source. In some embodiments, such conjugation produces controlled dynamic optical (e.g., fluorescent) point sources, wherein the diffusion of the optical (e.g., fluorescent) point sources is limited to the range of motion imposed by the conjugation of the polymer to the surface. In some embodiments, conjugation is performed via covalent bonding, adsorption, electrostatic bonding, hydrophobic bonding, or a combination thereof.

[0082] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a plurality of diffusing optical (e.g., fluorescent) point sources disposed within a polymer. In some embodiments, the polymer in which the plurality of diffusing optical (e.g., fluorescent) point sources are disposed is a hydrogel. In some embodiments, the hydrogel in which the plurality of diffusing optical (e.g., fluorescent) point sources are disposed is a polysaccharide hydrogel, polyacrylamide hydrogel, polyacrylic acid hydrogel, polymethyl methacrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or combinations thereof. In some embodiments, the hydrogel in which the plurality of diffusing optical (e.g., fluorescent) point sources are disposed is a polyacrylamide hydrogel or an agarose hydrogel. In some embodiments, the hydrogel in which multiple diffusible optical (e.g., fluorescent) point sources are disposed is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene (PTFE), polycarbonate, or polyvinyl butyral.

[0083] In some embodiments of this disclosure, the plurality of diffusing fluorescent dot sources comprise quantum dots. In some embodiments, the quantum dots have a maximum emission value of about 400 nm to about 720 nm. In some embodiments, the quantum dots have a maximum emission value of about 420 nm to about 480 nm. In some embodiments, the quantum dots have a maximum emission value of 450 nm. In some embodiments, the quantum dots have a maximum emission value of about 500 nm to about 550 nm. In some embodiments, the quantum dots have a maximum emission value of 525 nm. In some embodiments, the quantum dots have a maximum emission value of 545 nm. In some embodiments, the quantum dots have a maximum emission value of about 525 nm to about 575 nm. In some embodiments, the quantum dots have a maximum emission value of 565 nm. In some embodiments, the quantum dots have a maximum emission value of about 575 nm to about 650 nm. In some embodiments, the quantum dots have a maximum emission value of 585 nm. In some embodiments, the quantum dots have a maximum emission value of 605 nm. In some embodiments, the quantum dots have a maximum emission value of 625 nm. In some embodiments, the quantum dot has an emission maximum of about 650 nm to about 720 nm. In some embodiments, the quantum dot has an emission maximum of 655 nm. In some embodiments, the quantum dot has an emission maximum of 705 nm.

[0084] In some embodiments, the diffusible optical (e.g., fluorescent) point source comprises nanodiamond. In some embodiments, the nanodiamond has an emission maximum of about 400 nm to about 720 nm. In some embodiments, the nanodiamond has an emission maximum of about 400 nm to about 500 nm. In some embodiments, the nanodiamond has an emission maximum of about 415 nm. In some embodiments, the nanodiamond has an emission maximum of about 500 nm to about 550 nm. In some embodiments, the nanodiamond has an emission maximum of 510 nm. In some embodiments, the nanodiamond has an emission maximum of about 550 nm to about 600 nm. In some embodiments, the nanodiamond has an emission maximum of 575 nm. In some embodiments, the nanodiamond has an emission maximum of about 600 nm to about 700 nm. In some embodiments, the nanodiamond has an emission maximum of 638 nm.

[0085] 2.1.3. Reference sample containing uniform optical volume

[0086] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing a uniform optical (e.g., fluorescent) volume. For example, but not as a limitation, such uniform optical (e.g., fluorescent) volumes may be individually disposed within a matrix. In some embodiments, the matrix in which the uniform optical (e.g., fluorescent) volumes are disposed is a solution or a polymer. In some embodiments, the matrix in which the uniform optical (e.g., fluorescent) volumes are disposed is a solution comprising Tris:HCl, DMSO, DMEM, DPBS, or H2O. In some embodiments, the H2O is distilled H2O or deionized H2O. In some embodiments, the polymer matrix in which the uniform optical (e.g., fluorescent) volumes are disposed is a sol-gel, gel, or solid. In some embodiments, the polymer matrix in which the uniform optical (e.g., fluorescent) volumes are disposed is a hydrogel. In some embodiments, the hydrogel matrix in which a uniform optical (e.g., fluorescent) volume is disposed is a polysaccharide hydrogel, polyacrylamide hydrogel, polyacrylic acid hydrogel, polymethyl methacrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or a combination thereof. In some embodiments, the polymer matrix in which a uniform optical (e.g., fluorescent) volume is disposed is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene (PTFE), polycarbonate, or polyvinyl butyral.

[0087] In some embodiments, the uniform optical (e.g., fluorescent) volume comprises fluorescent molecules. Non-limiting examples of such fluorescent molecules include fluorescein, rhodamine 6G (R6G), and rhodamine B. In some embodiments, the fluorescent molecules are quenched fluorescent molecules. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 400 nm to about 720 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 420 nm to about 480 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of 450 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 500 nm to about 550 nm. For example, in some embodiments, the uniform fluorescence volume comprises fluorescein as a fluorescent molecule having an emission maximum of 517 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 525 nm to about 575 nm. For example, in some embodiments, the uniform fluorescence volume comprises rhodamine B as a fluorescent molecule having an emission maximum of 550 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 575 nm to about 650 nm. In some embodiments, the uniform fluorescence volume comprises fluorescent molecules having an emission maximum of about 650 nm to about 720 nm.

[0088] 2.1.4. Reference sample containing resolution test pattern

[0089] In some embodiments, this disclosure relates to compositions comprising a plurality of individually addressable reference samples, each containing one or more resolution test patterns. For example, but not as a limitation, such resolution test patterns may be absorptive or reflective. Exemplary resolution test patterns known in the art include, but are not limited to, the following patterns: USAF 1951 resolution test pattern; NBS 1952 resolution test pattern; Ronchi scribed target; and fan-shaped star target.

[0090] 2.2 Evaluation of the performance of instruments suitable for single-molecule tracking

[0091] 2.2.1 Calibration of instruments applicable to single-molecule tracking

[0092] In some embodiments, this disclosure relates to a method for evaluating multiple performance characteristics of an instrument suitable for tracking single molecules using a composition comprising a plurality of individually addressable reference samples. In some embodiments, the individually addressable reference samples used in the composition for evaluating the performance of an instrument suitable for tracking single molecules are selected from:

[0093] A reference sample containing multiple spatially stationary optical (e.g., fluorescent) point sources;

[0094] A reference sample containing multiple diffusible optical (e.g., fluorescent) point sources;

[0095] A reference sample containing a uniform optical (e.g., fluorescent) volume; and

[0096] A reference sample containing one or more resolution test patterns.

[0097] In some embodiments of such methods, the instrument is a microscope. In some embodiments, such methods include: aligning the objective lens of the microscope with one of a plurality of individually addressable reference samples, wherein the vertical axis of the objective lens is perpendicular to the horizontal axis of the sample; and

[0098] Analyze one or more imaging parameters based on images captured by a microscope;

[0099] Analyze one or more acquisition parameters based on images captured by a microscope;

[0100] Analyze one or more SMT analysis parameters based on images captured by a microscope; or

[0101] Its combination.

[0102] In some embodiments, the imaging parameters analyzed for the purposes of the methods disclosed herein include laser parameters, alignment parameters, camera parameters, detection parameters, and combinations thereof. For example, but not as a limitation, the analysis of laser parameters may include analyzing one or more of the following: laser wavelength, laser pulse, laser power, laser intensity, and laser pulse duration. In some embodiments, the analysis of alignment parameters may include analyzing one or both of the following: sheet characteristics and focal plane. In some embodiments, the analysis of sheet characteristics may include analyzing one or more of the following: sheet thickness; sheet uniformity; sheet tilt angle; and sheet intensity density. In some embodiments, the analysis of camera parameters may include analyzing one or more of the following: camera noise mode, relative spatial alignment, relative time synchronization, orientation, or magnification. In some embodiments, the analysis of detection parameters may include analyzing one or more of the following: aberrations, sensitivity, and resolution. In some embodiments, the analysis of aberrations may include analyzing one or more of the following: spherical aberration, chromatic aberration, coma aberration, and cloverleaf aberration. In some embodiments, analyzing the focal plane includes analyzing the position, flatness, orientation relative to the sample and detector, and thickness of the optical focal plane.

[0103] In some embodiments, the acquisition parameters analyzed for the purposes of the methods disclosed herein may include analyzing temporal and / or spatial parameters. In some embodiments, the analysis of temporal parameters may include analyzing one or more of frame rate, exposure time, number of frames, and channels. In some embodiments, the analysis of spatial parameters may include analyzing one or more of the field of view (FOV) size on the detector chip, the FOV position on the detector chip, and the FOV position in the sample.

[0104] In some embodiments, the SMT parameters analyzed for the purposes of the methods disclosed herein may include analyzing single-molecule localization and / or single-molecule tracing. In some embodiments, the analysis of single-molecule localization may include analyzing one or more of localization error, spot count, or signal-to-noise ratio (SNR). In some embodiments, the analysis of single-molecule tracing may include analyzing one or more of trajectory count, trajectory length, jump length, and mean posterior diffusion coefficient.

[0105] In some embodiments, the performance characteristic analyzed with respect to one or more of the performance parameters described herein is excitation flux. In some embodiments, excitation flux is analyzed by determining the intensity of the fluorescent dye. In some embodiments, excitation flux is analyzed by determining the intensity of one or more point sources. In some embodiments, excitation flux is analyzed by determining the thickness of the light sheet.

[0106] In some embodiments, the performance characteristics analyzed with respect to one or more of the performance parameters described herein are selected from: illumination module rotation, e.g., OLS rotation; rotation relative to the camera; camera clock synchronization; illumination module angle; illumination module fringes, e.g., OLS fringes; and illumination uniformity, e.g., OLS uniformity. For example, but not as a limitation, the relative orientation of one or more resolution test patterns (e.g., Ronchi grating patterns) can be used to facilitate the determination of such performance characteristics. In some embodiments, the resolution test pattern can be aligned to a support that is itself aligned to the sample stage, and the camera can be configured to visualize the resolution test pattern. The system can then be adjusted (e.g., rotated) to minimize the angle, thereby synchronizing the camera with the sample stage clock. In some embodiments, the relative orientation of the stationary light sheet (i.e., the case where the light sheet is not scanned) can be determined when the fluorescence (when using a fluorescent dye) appears as a line. Then, using a camera that has been clocked synchronized, the illumination module can be rotated to minimize the angle between excitation and detection, thereby synchronizing the illumination module with the camera clock.

[0107] In some embodiments, the performance characteristics analyzed with respect to one or more of the performance parameters described herein are plate position and levelness. For example, but not as a limitation, the system described herein may employ a Perfect Focus System (PFS) unit (NIKON). ®Alternatively, a similar unit can be used to send a near-infrared laser beam to the bottom of the glass of the sample holder, where the back reflection of the beam can be projected onto a position-sensitive support. Such projection is advantageous for detecting the positioning of the focal plane relative to the bottom surface of the sample holder glass (e.g., 1 μm into the sample). In some embodiments, this allows the focus to be locked onto the sample, and the objective lens will automatically follow the height and curvature of the sample holder.

[0108] In some embodiments, the performance characteristic analyzed with respect to one or more of the performance parameters described herein is field-of-view (FOV) uniformity. In some embodiments, FOV uniformity can be determined by monitoring the fluorescence of pure and homogeneous dye solutions. For example, but not as a limitation, such analysis of pure and homogeneous dye solutions allows characterizing the uniformity of the illumination module in SMT imaging. In some embodiments, monitoring SMT parameters across FOV (e.g., as described herein) allows characterizing the uniformity of single-molecule detection capability.

[0109] In some embodiments, the performance characteristic analyzed with respect to one or more of the performance parameters described herein is the tilt angle. For example, in some embodiments, the light sheet may be positioned at the center of the FOV, and the light sheet is sent into a sample containing a thin layer of point source attached to the bottom of the aperture (e.g., a PAA containing Qdot). In such embodiments, the microscope may perform z-stacking, which results in direct monitoring of the 3D shape of the light sheet, and subsequent analysis for the location of extracted fluorescence intensity allows the calculation of the tilt angle.

[0110] In some embodiments, the performance characteristics analyzed with respect to one or more of the performance parameters described herein are background and / or camera noise. For example, in some embodiments, different types of noise are recorded, for example, with or without a sample and / or with or without a laser, allowing comparisons to determine the noise in the test sample. Regarding SNR analysis, this can be accomplished using a single-molecule localization algorithm and by calculating SMT parameters.

[0111] In some embodiments, the performance characteristics analyzed with respect to one or more of the performance parameters described herein are mechanical and / or temperature effects. For example, in some embodiments, longitudinal monitoring is used to check alignment and SMT parameters with or without alteration to the system (e.g., mechanical perturbations can cause changes in the relative orientation of the sheet to the camera and will indicate mechanical instability of certain components such as the camera mount). In some embodiments, thermal perturbations can be detected because they can cause increased molecular diffusion due to increased Brownian motion or the introduction of optical aberrations.

[0112] In some embodiments, the performance characteristic analyzed with respect to one or more of the performance parameters described herein is the point spread function (PSF). For example, but not as a limitation, this can be determined by imaging point sources (e.g., single or multiple, and then combined) in 2D or 3D, and by characterizing their spatial extent, symmetry, and intensity. The PSF is then typically analyzed / evaluated by Strell ratio, FWHM, and aberrations (e.g., spherical aberration, coma aberration, cloverleaf aberration, astigmatism).

[0113] In some embodiments, the performance characteristic analyzed with respect to one or more of the performance parameters described herein is a correction loop. For example, but not as a limitation, the correction loop primarily affects spherical aberration, and the correction loop for a given sample and imaging conditions can be monitored as a subset of the PSF characterization. In some embodiments, the quality factor can be, but is not limited to, PSF parameters (e.g., FWHM, symmetry, and intensity) and / or SMT measures (SNR and localization error).

[0114] In some embodiments, the performance characteristic analyzed with respect to one or more of the performance parameters described herein is the focal plane. For example, in some embodiments, a scanning sheet is used across a sample containing a thin layer of point sources attached to the bottom of the aperture (e.g., a PAA containing Qdots). In these embodiments, the microscope can perform z-stacking, which results in direct monitoring of the focal plane, and subsequent analysis for the sharpness of the extracted point sources allows for the calculation of the focal plane's position, orientation, and thickness.

[0115] In some embodiments, the performance characteristic analyzed with respect to one or more of the performance parameters described herein is the focal plane. For example, but not as a limitation, the position, orientation, and flatness of the focal plane for a given microscope alignment can be monitored as a subset of the imaging sharpness. In some embodiments, the quality factor may be, but is not limited to, Volath (i.e., a focus metric that measures image sharpness by calculating the sum of absolute differences between adjacent pixels, thereby capturing edge intensity), Tenengrad (i.e., a gradient-based focus metric that evaluates image sharpness by calculating the magnitude of gradients using the Sopper operator to emphasize high-frequency content), Redondo (i.e., a focus metric that estimates image sharpness by measuring the spread or width of the image energy distribution using a Laplacian-based operator), Laplacian variance (i.e., a focus metric that quantifies image sharpness by calculating the variance of the Laplacian operator of the image, thereby emphasizing regions with high contrast or rapid intensity changes), FFTBP (Fast Fourier Transform Bandpass) (i.e., a frequency domain focus metric that evaluates image sharpness by analyzing the magnitude of high-frequency components after applying a bandpass filter to the Fourier-transformed image), or Fourier ring correlation (FRC) (i.e., a frequency domain focus metric that quantifies image sharpness by measuring the spatial frequency correlation between two independent images, thereby indicating the level of detail preserved across images). For example, but not as a limitation, the results of focal plane characterization can be obtained through the Perfect Focus System (PFS) unit (NIKON). ® Or similar units that can be locked onto the surface of the bottom glass of the sample holder and facilitate the detection of the focal plane relative to the surface of the bottom glass of the sample holder (e.g., 1 μm into the sample) can be integrated manually or automatically.

[0116] 2.2.2 Integrated evaluation of instruments suitable for single-molecule tracking

[0117] In some embodiments, this disclosure relates to methods for evaluating multiple performance characteristics of an instrument suitable for tracking single molecules, wherein the methods further include repeated alignment and analysis steps for multiple individually addressable reference samples, and integrating the analyses performed on the multiple individually addressable reference samples, thereby evaluating multiple microscope performance characteristics. For example, but not as a limitation, such integration can take the form of calculating a comprehensive score based on the performance characteristic evaluation. In some embodiments, instrument modifications are performed manually in response to the results of the integrated evaluation. In some embodiments, instrument modifications are performed automatically in response to the results of the integrated evaluation. In some embodiments, both manual and automatic modifications to the instrument are performed in response to the results of the integrated evaluation.

[0118] In some embodiments, the performance characteristics analyzed with respect to one or more performance parameters and integrated into an analysis performed on multiple individually addressable reference samples are selected from: illumination module rotation, e.g., OLS rotation; rotation relative to the camera; camera clock synchronization; illumination module angle; illumination module fringes, e.g., OLS fringes; and illumination uniformity, e.g., OLS uniformity. For example, but not as a limitation, the relative orientation of one or more resolution test patterns (e.g., Ronchi grating patterns) can be used to facilitate the determination of such performance characteristics. In some embodiments, the resolution test pattern can be aligned to a support that is itself aligned to the sample stage, and the camera can be configured to visualize the resolution test pattern. The system can then be adjusted (e.g., rotated) to minimize the angle, thereby synchronizing the camera with the sample stage clock. In some embodiments, the relative orientation of the stationary light sheet (i.e., the case where the light sheet is not scanned) can be determined when the fluorescence (when using a fluorescent dye) appears as a line. Then, using a camera that has been clocked synchronized, the illumination module can be rotated to minimize the angle between excitation and detection, thereby synchronizing the illumination module with the camera clock.

[0119] In some embodiments, the performance characteristics analyzed with respect to one or more of the performance parameters and integrated into the analysis performed on multiple individually addressable reference samples are plate position and levelness. For example, but not as a limitation, the system described herein may employ a Perfect Focus System (PFS) unit (NIKON). ® Alternatively, a similar unit can be used to send a near-infrared laser beam to the bottom of the glass of the sample holder, where the back reflection of the beam can be projected onto a position-sensitive support. Such projection is advantageous for detecting the positioning of the focal plane relative to the bottom surface of the sample holder glass (e.g., 1 μm into the sample). In some embodiments, this allows the focus to be locked onto the sample, and the objective lens will automatically follow the height and curvature of the sample holder.

[0120] In some embodiments, the performance characteristic analyzed in relation to one or more performance parameters and integrated into analyses performed on multiple individually addressable reference samples is field-of-view (FOV) uniformity. In some embodiments, FOV uniformity can be determined by monitoring the fluorescence of pure and homogeneous dye solutions. For example, but not as a limitation, such analysis of pure and homogeneous dye solutions allows characterizing the uniformity of the illumination module in SMT imaging. In some embodiments, monitoring SMT parameters across the FOV (e.g., as described herein) allows characterizing the uniformity of single-molecule detection capability.

[0121] In some embodiments, the performance characteristic analyzed for one or more of the performance parameters and integrated into analyses performed on multiple individually addressable reference samples is the tilt angle. For example, in some embodiments, the light sheet may be positioned at the center of the FOV, and the light sheet is sent to a sample containing a thin layer of point source attached to the bottom of the aperture (e.g., a PAA containing Qdot). In such embodiments, the microscope may perform z-stacking, which results in direct monitoring of the 3D shape of the light sheet, and subsequent analysis for the location of extracted fluorescence intensity allows the tilt angle to be calculated.

[0122] In some embodiments, the performance characteristics analyzed in terms of one or more performance parameters and integrated into analyses performed on multiple individually addressable reference samples are background and / or camera noise. For example, in some embodiments, different types of noise are recorded, for example, with or without a sample and / or with or without a laser, allowing comparisons to determine the noise in the test sample. Regarding SNR analysis, this can be accomplished using a single-molecule localization algorithm and by calculating SMT parameters.

[0123] In some embodiments, the performance characteristics analyzed in relation to one or more performance parameters and integrated into analyses performed on multiple individually addressable reference samples are mechanical and / or temperature effects. For example, in some embodiments, longitudinal monitoring is used to check alignment and SMT parameters with or without alteration to the system (e.g., mechanical perturbations can cause changes in the relative orientation of the sheet to the camera and will indicate mechanical instability in certain components, such as the camera mount). In some embodiments, thermal perturbations can be detected because they can cause increased molecular diffusion due to increased Brownian motion or the introduction of optical aberrations.

[0124] In some embodiments, the performance characteristic analyzed in terms of one or more performance parameters and integrated into an analysis performed on multiple individually addressable reference samples is the point spread function (PSF). For example, but not as a limitation, this can be determined by imaging point sources (e.g., single or multiple, then combined) in 2D or 3D, and characterizing their spatial extent, symmetry, and intensity. The PSF is then typically analyzed / evaluated by Strell ratio, FWHM, and aberrations (e.g., spherical aberration, coma, cloverleaf aberration, astigmatism). In some embodiments, analyzing the focal plane includes analyzing the position, flatness, orientation relative to the sample and detector, and thickness of the optical focal plane.

[0125] In some embodiments, the performance characteristic analyzed with respect to one or more performance parameters and integrated into an analysis performed on multiple individually addressable reference samples is a correction loop. For example, but not as a limitation, the correction loop primarily affects spherical aberration, and the correction loop for a given sample and imaging conditions can be monitored as a subset of the PSF characterization. In some embodiments, the quality factor can be, but is not limited to, PSF parameters (e.g., FWHM, symmetry, and intensity) and / or SMT metrics (SNR and localization error).

[0126] In some embodiments, the performance characteristic analyzed with respect to one or more of the performance parameters described herein is the focal plane. For example, in some embodiments, a scanning sheet is used across a sample containing a thin layer of point sources attached to the bottom of the aperture (e.g., a PAA containing Qdots). In these embodiments, the microscope can perform z-stacking, which results in direct monitoring of the focal plane, and subsequent analysis for the sharpness of the extracted point sources allows for the calculation of the focal plane's position, orientation, and thickness.

[0127] In some embodiments, the performance characteristic analyzed with respect to one or more performance parameters and integrated into an analysis performed on multiple individually addressable reference samples is the focal plane. For example, but not as a limitation, the position, orientation, and flatness of the focal plane for a given microscope alignment can be monitored as a subset of imaging sharpness. In some embodiments, the quality factor may be, but is not limited to, Volath (i.e., a focus metric that measures image sharpness by calculating the sum of absolute differences between adjacent pixels, thereby capturing edge intensity), Tenengrad (i.e., a gradient-based focus metric that evaluates image sharpness by calculating the magnitude of gradients using the Sopper operator to emphasize high-frequency content), Redondo (i.e., a focus metric that estimates image sharpness by measuring the spread or width of the image energy distribution using a Laplacian-based operator), Laplacian variance (i.e., a focus metric that quantifies image sharpness by calculating the variance of the Laplacian operator of the image, thereby emphasizing regions with high contrast or rapid intensity changes), FFTBP (Fast Fourier Transform Bandpass) (i.e., a frequency domain focus metric that evaluates image sharpness by analyzing the magnitude of high-frequency components after applying a bandpass filter to the Fourier-transformed image), or Fourier ring correlation (FRC) (i.e., a frequency domain focus metric that quantifies image sharpness by measuring the spatial frequency correlation between two independent images, thereby indicating the level of detail preserved across images). For example, but not as a limitation, the results of focal plane characterization can be obtained through the Perfect Focus System (PFS) unit (NIKON). ® Or similar units that can be locked onto the surface of the bottom glass of the sample holder and facilitate the detection of the focal plane relative to the surface of the bottom glass of the sample holder (e.g., 1 μm into the sample) can be integrated manually or automatically.

[0128] 3. Exemplary single-molecule tracking hardware

[0129] 3.1. Image Acquisition System

[0130] refer to Figure 3 Aspects of the current topic can be implemented using an SMT workflow, where such a workflow incorporates a system for image acquisition. For example, such image acquisition can be combined with imaging of a sample to generate a series of images and / or videos. In some embodiments, an exemplary image acquisition system includes: a light source configured to emit light relayed by one or more optical elements in an optical repeater configured to shape the light emitted from the light source to form a shaped beam such that the shaped beam has uniform intensity along a longer dimension of a linear shape; optical elements, such as a galvanometer, configured to translate the shaped beam and may be positioned before or after the optical repeater configured to form the shaped beam such that the shaped beam has uniform intensity along a longer dimension of a linear shape; and one or more optical elements, such as a dichroic mirror, configured to guide the shaped beam to an objective lens, whereby a portion of the sample plane is illuminated by the tilted beam, thereby producing emission of light (e.g., fluorescence emission) from the sample, which is focused by the objective lens through a series of optical elements (e.g., lenses and emission filters) to the image acquisition system.

[0131] In some embodiments, a microscope system for performing the methods of this disclosure may include (a) a stage for supporting a cell-free sample, wherein the cell-free sample contains a target molecule, such as a fluorescent molecule; (b) a light source for emitting a light beam capable of inducing a light-based response from the target molecule (e.g., a fluorescent molecule) in the cell-free sample; (c) an objective lens for focusing the light beam onto the cell-free sample in a sample plane, wherein the target molecule (e.g., a fluorescent molecule) in the sample is positioned in the field of view in the sample plane; (d) a detector device for monitoring the light-based response from the target molecule (e.g., a fluorescent molecule) over a period of time (e.g., in the presence of a test molecule); (e) a memory; and (f) a processor communicating with the memory and the detector device. In some embodiments, the processor is capable of determining the diffusion coefficient of the target molecule (e.g., a fluorescent molecule) and comparing it with a reference diffusion coefficient. In some embodiments, the processor is capable of determining the diffusion coefficient of the target molecule (e.g., a fluorescent molecule) in the presence of a test molecule. In some embodiments, the reference diffusion coefficient is the diffusion coefficient of the target molecule (e.g., a fluorescent molecule) in the absence of a test molecule.

[0132] Figure 4A A schematic diagram of an exemplary image acquisition system of this disclosure is depicted, wherein the XZ plane is visible. Figure 4BThe same exemplary image acquisition system is depicted, but the YZ plane is visible. In a particular embodiment, the exemplary illumination and image acquisition system (2-001) may include: a light source (2-005) configured to emit light; a scanning element (2-022) (e.g., a galvanometer) configured to selectively scan the beam to translate the beam at a sample plane (2-130); an optical shaping element (2-025) configured to receive the beam from the scanning element and generate a beam having an elongated and linear cross-sectional shape; and an optical repeater (2-045) configured to guide the beam to an objective lens (2-120), whereby a portion of the sample plane (2-130) is illuminated by the tilted beam (2-125), such that light emission (e.g., fluorescence emission) from the sample is focused by the objective lens (2-120) through a series of optical elements (e.g., a tube lens (2-155) and one or more emission filters (2-150, 2-160)) to an image collection system (2-165).

[0133] 3.1.1. Light source

[0134] refer to Figure 4A An exemplary image acquisition system is provided, comprising a light source (2-005) configured to emit light. In some embodiments of the image acquisition system disclosed herein, the light source (2-005) may be configured to emit light of a single wavelength. In some embodiments of the image acquisition system disclosed herein, the light source (2-005) may be configured to emit light of two, three, four, five, or more individual wavelengths. In some embodiments, the wavelength of the light emitted by the light source is predetermined. For example, but not as a limitation, the wavelength may be predetermined such that the emitted light induces fluorescence emission when illuminating a sample (e.g., a sample containing a fluorescent protein). In some instances, the wavelength used in conjunction with the methods described herein will fall within the range of 400 nm to 650 nm. In some instances, the light source (2-005) will emit light with wavelengths between 400 nm and 408 nm, between 550 nm and 565 nm, or between 638 nm and 650 nm. In some non-limiting embodiments, the light source (2-005) is configured to include three lasers having nominal center wavelengths of 405 nm, 560 nm, and 640 nm, which can vary within the absorption band of the fluorophore used. In some instances, a 405 nm wavelength is used to excite the Hoechst dye. In some instances, a 560 nm wavelength is used to excite the dye attached to HaloTag® (e.g., JF549). In some instances, a 640 nm wavelength is used to excite the dye (e.g., Potomac Red).

[0135] In some non-limiting embodiments, the light source (2-005) is used to catalyze a photochemical reaction. For example, but not as a limitation, the wavelength and illumination intensity can cause the breaking of chemical bonds. As another example, but not as a limitation, the wavelength and illumination intensity can induce the adoption of a nonradiative dark state (i.e., "photobleaching of molecules"). As another example, but not as a limitation, the wavelength and illumination intensity can induce radiative or nonradiative energy transfer between fluorophores within the sample.

[0136] In some embodiments of the image acquisition system described herein, the light source (2-005) may be configured to deliver a predetermined amount of power to the back focal plane of the objective lens (2-105). For example, but not by limitation, the light source (2-005) delivers greater than 10 mW for some wavelengths (e.g., 405 nm) and / or greater than 150 mW for other wavelengths (e.g., 640 nm). Alternatively or additionally, in cases where the light source (2-005) comprises three lasers emitting at wavelengths of 405 nm, 560 nm, and 640 nm, respectively, the light source (2-005) may be configured to deliver a predetermined amount of power to the back focal plane of the objective lens (2-105). For example, but not by limitation, 405 nm may be configured to provide >10 mW; 560 nm may be configured to deliver >150 mW; and 640 nm may be configured to deliver >150 mW.

[0137] In some embodiments of the image acquisition system described herein, the light source (2-005) is configured to emit pulsed light. For example, but not as a limitation, the light source (2-005) may be configured to emit radio-flicker pulsed light. In some embodiments of the image acquisition system described herein, the light source (2-005) is configured to emit pulsed light synchronously with the start of image acquisition. In some non-limiting embodiments, the light source (2-005) pulses at specific time intervals according to the number of frames captured per second. For example, but not as a limitation, if the detector (2-165) is capturing 100 frames per second (FPS), the laser is turned on for 9 ms and off for 1 ms. Conversely, in 200 FPS mode, the laser is turned on for 4 ms and off for 1 ms. In some embodiments of the OLS htSMT workflow, the light source is configured to reduce power from 90% to 10% in less than about 0.4 ms. In some embodiments of the OLS htSMT workflow, the light source is configured to reduce power from 90% to 10% in less than about 0.2 ms.

[0138] In some embodiments, the emission of light from the light source (2-005) and the guidance of that light into the image acquisition system disclosed herein can be facilitated using single-mode optical fiber. Alternatively, multimode optical fiber can be employed in some embodiments of the image acquisition system disclosed herein. For example, but not as a limitation, the multimode optical fiber can be configured in a predetermined shape for sample illumination.

[0139] In some embodiments of the image acquisition system described herein, such as for systems configured for high-throughput sample analysis, the light source (2-005) can be configured to exhibit low drift in power output. In some embodiments, such a low-drift configuration increases sample processing consistency to facilitate high-throughput analysis. For example, but not by limitation, such a low-drift power output configuration maintains the power output within a variation of about 0% to about 15%, about 0% to about 10%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%.

[0140] In some instances, such low-drift power output is configured to maintain power output within approximately 0% to approximately 15%, approximately 0% to approximately 10%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1% variation under ambient (room) temperature variations, such as 17°C + / - 5°C. In some instances, this is achieved by using temperature sensors and / or closed-loop heaters to maintain a stable temperature of the internal light source (e.g., laser engine), thereby reducing output power drift. For example, but not as a limitation, the light source can use an insulated housing design to thermally insulate against ambient temperature fluctuations. Alternatively or additionally, the closed-loop heater can be strategically placed at specific locations in the system (e.g., fiber couplers) to reduce output drift. Alternatively or additionally, water jackets and / or coolers can be used to reduce heat buildup from the laser head. Furthermore, these thermal controls, used individually or in combination, shorten the heating time to reach steady-state operation and maintain a more stable internal operating temperature when the laser is powered off and on.

[0141] 3.1.2. Sample Illumination

[0142] In some embodiments, the system of this disclosure includes a light source configured to emit light relayed by one or more optical elements in an optical repeater, the optical repeater being configured to shape the light emitted from the light source to form a shaped beam. Specific optical elements of any particular optical repeater implementation can be selected and configured to generate a suitably shaped beam and to provide a suitable translation of the beam.

[0143] In some non-limiting embodiments of the optical repeater in the currently disclosed image acquisition system, the optical repeater will include one or more lenses and / or other optical elements. For example, but not as a limitation, the selection and orientation of the lenses and other optical elements in the optical repeater will be configured to properly shape the beam guided to the sample. In some non-limiting embodiments, the optical repeater will include optical elements for collimating the emitted light from the light source, such as collimators. Additionally or alternatively, the optical repeater will include additional optical elements, such as Powell lenses or other elements suitable for generating a fan-shaped beam, one or more cylindrical lenses, one or more slits for adjusting the beam range, one or more achromatic lenses and / or one or more mirrors, one or more of which may be galvanometers capable of translating light. Specific properties of the optical elements will be predetermined to produce a properly shaped beam. For example, but not as a limitation, the SMT system of this disclosure can achieve a uniform horizontal FOV as well as a uniform vertical FOV. Such uniformity in the horizontal and vertical FOV contrasts with other strategies that provide a non-uniform horizontal FOV and / or a non-uniform vertical FOV.

[0144] To achieve uniform horizontal and vertical FOV, the optical repeater of the SMT system described herein includes optical elements or components capable of generating a beam that extends along the X-plane and narrows along the Y-plane, wherein the beam has uniform intensity over the longer dimension of a linear shape. In some non-limiting implementations, the optical repeater of the SMT system described herein will include a Powell lens to shape the beam such that it has uniform intensity over the longer dimension of a linear shape. The optical repeater of the SMT system described herein may include additional or alternative optical elements or components to shape the beam such that it has uniform intensity over the longer dimension of a linear shape. For example, but not as a limitation, the optical repeater of the SMT system described herein may include diffractive elements or components configured to shape the beam such that it has uniform intensity over the longer dimension of a linear shape.

[0145] In some non-limiting embodiments of optical repeaters in currently disclosed image acquisition systems, the optical repeater will include one or more optical elements or components configured to translate a beam of light relative to the sample plane of the sample to be analyzed, for example, translating the beam in a direction orthogonal to the longer dimension of the beam. For example, but not as a limitation, such optical elements or components configured to translate the beam of light relative to the sample plane of the sample to be analyzed may include galvanometers or piezoelectric elements configured to translate the beam. Additionally or alternatively, such optical elements or components configured to translate the beam of light relative to the sample plane of the sample to be analyzed may include computer-controlled motors. In some embodiments, the optical elements or components configured to translate the beam may be positioned before or after an optical element or component, such as a Powell lens, configured to shape the beam such that it has uniform intensity along the longer dimension of a linear shape.

[0146] In some embodiments, the system includes an optical repeater configured to shape light emitted from a light source to form a shaped beam, which is then guided by an optical element (e.g., a dichroic mirror) configured to direct the shaped beam to an objective lens, thereby illuminating the sample plane with the tilted beam.

[0147] refer to Figures 4A to 4B An exemplary image acquisition system is schematically illustrated, comprising a light source (2-005) configured to emit light. In a particular embodiment, the light source (2-005) (such as a fluorescent excitation light source) may provide a light beam to a pre-adjusted optical element or assembly (2-020), which may be configured to provide a collimated light beam to other optical elements and assemblies. Figures 4A to 4B and Figures 5A to 5E The schematic, non-limiting example shown includes a collimator (2-020).

[0148] In a particular embodiment, the scanning element (2-022) may be configured to receive a collimated beam (such as from a pre-adjusted optical assembly (2-020)) and selectively scan the received collimated beam. As a non-limiting example, the scanning element (2-022) may include one or more galvanometers, one-dimensional galvanometers, polygonal mirrors or scanners, micromirrors, microelectromechanical (MEMS) scanning mirrors, optical modulators and / or piezoelectric elements. Additionally or alternatively, such optical elements or assemblies configured to translate the beam may include one or more actuators and / or motors and may be computer-controlled. In a particular embodiment, the collimated beam may immediately have a Gaussian and / or circular cross-sectional profile after being intercepted by the scanning element (2-022), such as as a non-limiting example in… Figure 4A The diagram is schematically shown in (2-015). Figures 4A to 4B and Figures 5A to 5EThe schematic, non-limiting example shown includes a galvanometer (2-022), which in Figures 4A to 4B , Figure 5B and Figure 5D It is clearly described in the text.

[0149] In some embodiments, the optical shaping element (2-025) can be positioned to receive a light beam from the scanning element (2-022) and generate a linear beam, i.e., a beam having an elongated, linear, and / or linear cross-sectional shape. As a non-limiting example, the linear beam may extend along the X direction and narrow along the Y direction in the XY sample plane, such as at least in… Figure 4B As shown in the illustration.

[0150] By way of example and not limitation, the optical shaping element (2-025) may include one or more of a Powell lens, a diffractive optical element, an optical line generator, and / or a gradient refractive index (GRIN) optical element. Figures 4A to 4B and Figures 5A to 5E The schematic, non-limiting example shown includes a Powell lens as an optical shaping element (2-025), the vertex of which (2-028) is at least in Figure 4A and Figure 5C As shown in the image.

[0151] In certain embodiments, the linear beam formed by the optical shaping element (2-025) can be uniform, either alone or additionally, i.e., have uniform intensity along the longer dimension of its elongated and linear shape. In certain embodiments, the linear beam formed by the optical shaping element (2-025) can be collimated, either alone or additionally, along one or more directions. In some other embodiments, the optical shaping element (2-025) can provide and cooperate with an optical repeater (2-045) such that the combination of (2-025) and (2-045) can produce a collimated and linear beam having an elongated, linear, and / or linear cross-sectional shape, and uniform intensity along the longer dimension of its elongated and linear shape.

[0152] In certain embodiments, the optical repeater (2-045) may be configured to collimate, manipulate, filter, crop, stretch, modulate, modify, reshape, adjust, and / or redirect part or all of a light beam (such as a linear beam) received from the optical shaping element (2-025). Alternatively or additionally, in certain embodiments, the optical repeater (2-045) may be configured to focus a collimated and linear light beam at the back focal plane (2-105) of the objective lens (2-120). In some non-limiting embodiments, the optical repeater (2-045) may include one or more lenses, such as achromatic lenses, doublet lenses, slits, adjustment mechanisms, and / or other optical elements and components suitable for the purposes and functions disclosed above.

[0153] For example, but not as a limitation, based on the interoperability of at least scanning elements (such as galvanometers (2-022)), optical shaping elements (2-025) (such as Powell lenses), and / or optical repeaters (2-045), the OLS htSMT system of this disclosure can achieve a uniform horizontal FOV and a uniform vertical FOV. Such uniformity in the horizontal and vertical FOV can contrast with other strategies that can provide a non-uniform horizontal FOV and / or a non-uniform vertical FOV. In a particular embodiment, the horizontal uniformity described herein may refer to the uniformity of sample illumination light intensity, image light intensity, or pixel values ​​and / or signal-to-noise ratio (SNR). In a particular embodiment, the vertical uniformity described herein may refer to the uniformity of sample illumination light intensity, image light intensity, or pixel values ​​and / or signal-to-noise ratio (SNR).

[0154] Figures 4A to 4B and Figures 5A to 5E The schematic, non-limiting examples shown include: a scanning lens (2-050) configured to collect and / or collimate a beam of light received from a Powell lens acting as an optical shaping element (2-025); a slit (2-055) configured to limit and / or otherwise filter non-uniform aspects of the received beam, such as at the edges; and a tube lens (2-090) configured to guide and / or focus a linear beam of light at the back focal plane (2-105) of the objective lens (2-120). In particular embodiments, the scanning lens (2-050) and / or the tube lens (2-090) may include one or more achromatic lenses. As a further illustration and not a limitation, the cross-sectional shape (2-030) of the linear beam of light received from the Powell lens acting as an optical shaping element (2-025) may initially include specific features such as a bright nucleus (2-035) and / or edge non-uniformity (2-040). One or more elements of the optical repeater (2-045) can be configured such that the cross-sectional shape of the linear beam can be formed into a uniform, linear, and elongated shape, as shown by the beam shaping (2-065).

[0155] According to a specific embodiment, one or more elements of the optical repeater (2-045) can focus a linear beam at the rear focal plane (2-105) of the objective lens (2-120). One or more additional optical elements or components (2-100), such as a dichroic mirror, can be used to selectively route the linear beam toward the objective lens (2-120).

[0156] like Figures 4A to 4DAs shown in the non-limiting example, the objective lens (2-120) can be configured to tilt the light beam relative to the optical axis of the objective lens (2-120) by an angle (φ), and to illuminate the sample plane (2-130) by projecting and / or focusing the light beam at the sample plane (2-130). In a particular embodiment, the tilt angle (φ) can be set in the range of 30°-73°. In a particular embodiment, the tilt angle (φ) can be set in the range of 55°±10° to 63°±10°. In a particular embodiment, the tilt angle (φ) can be set in the range of 57°±5° to 60°±5°. In a particular embodiment, the tilt angle (φ) can be 53°, 54°, 55°, 56°, 57°, 58°, 59°, 60°, 61°, 62°, or 63°.

[0157] Individually or additionally, the tilt angle (φ) can be varied or optimized based on sample parameters, stacking tolerances, and / or microscope parameters, including aspects along the optical path further than the dichroic mirror in the assembly, or aspects further than the sample illumination. Therefore, in a particular embodiment, the tilt angle (φ) can be varied individually or additionally based on the factors described above.

[0158] As a non-limiting example, to provide a highly tilted linear beam to the objective lens (2-120), the linear beam can be introduced into the objective lens (2-120) such that it deviates from the optical axis of the objective lens (2-120). As a non-limiting example, in a particular embodiment, the linear beam may deviate from the optical axis of the objective lens (2-120) by 3.6 mm to 4 mm, or according to a particular embodiment, by 2.5 mm to 4 mm. In some embodiments, the linear beam may deviate from the optical axis of the objective lens (2-120) by 2.5 mm, 2.75 mm, 3 mm, 3.25 mm, 3.5 mm, 3.75 mm, or 4 mm.

[0159] Alone or additionally, the offset may be changed or optimized based on stacking tolerances and / or variable microscope parameters, including aspects along the optical path further than the dichroic mirror in the assembly, or aspects further than the sample illumination; in certain embodiments, the offset may be further varied accordingly.

[0160] Figure 4B A view (2-110) of the back focal plane (2-105) is shown, as seen by an observer traveling with the illumination beam and entering the objective lens (2-120). The appearance of the beam in the cross-section of the back focal plane is schematically indicated by (2-115). An illuminated view (2-135) of the sample plane (2-130) is also shown. Figure 4BAs seen by an observer positioned on the optical axis of objective lens (2-120) and receiving the illumination beam (i.e., observing toward objective lens (2-120)), where (2-140) schematically shows the appearance of a linear and uniform beam of light at the sample plane.

[0161] As discussed earlier in this document, in certain embodiments, scanning elements (such as a galvanometer (2-022)) can selectively scan a collimated beam, such that a linear, uniform beam (2-140) can be scanned, traversed, or translated accordingly in the sample plane (2-130). In certain embodiments, such selective scanning of the scanning element (2-022) can cause the linear, uniform beam in the sample plane (2-130) to be translated along a direction orthogonal to the longer dimension of the elongated and linear cross-sectional shape of the beam. As a non-limiting example, at least Figure 4B , Figure 4C and Figure 5A The specific linear and / or rotating double arrow symbols in the diagram schematically indicate the scanning direction of the corresponding optical element, module, and / or beam.

[0162] refer to Figures 4A to 4D and Figures 5A to 5E The non-limiting example shown in the figure has a galvanometer (2-022) positioned around a pivot (e.g., Figure 4B and Figure 5D Selective rotation of 2-024 in the image allows the collimated beam (such as 2-015) to be scanned along the vertex (2-028) of the Powell lens, which acts as an optical shaping element (2-025). In a particular embodiment, the collimated beam received at the vertex (2-028) of the Powell lens, which acts as an optical shaping element (2-025), can remain collimated after being truncated and subsequently exiting from the Powell lens, which acts as an optical shaping element (2-025), despite multiple refractions occurring therein. Accordingly, the linear, uniform beam at the sample plane (2-130) can be selectively translated in a direction orthogonal to the longer dimension of the beam. Continuing to refer to Figures 4A to 4D and Figures 5A to 5E In the non-limiting example shown, a linear, uniform beam (2-140) in the XY sample plane (2-130) formed by a tilted beam (2-125) passing through the sample plane can be selectively translated or traversed along the Y direction based on selective scanning of the galvanometer (2-022), as indicated.

[0163] In some non-limiting embodiments of the image acquisition system of this disclosure, the objective (2-120) can guide a tilted beam (2-125) onto the sample plane (2-130) to be analyzed. In some non-limiting embodiments of the image acquisition system of this disclosure, the objective (2-120) can be a water immersion objective. The use of a water immersion objective facilitates high-throughput sample analysis by eliminating the oil present as associated with the use of an oil immersion objective, thereby achieving higher image quality and less distortion. Not only does the presence of oil create problems in the environment of automated systems (where oil can spread to components, including optical elements that may be contaminated by exposure to oil), but water immersion objectives also have a better exponential match for imaging cells, resulting in less distortion and therefore higher image quality than oil immersion objectives. In some non-limiting embodiments, the objective is a 60 x 1.27 NA water immersion objective (Nikon). In some embodiments of the workflow described herein, the water immersion objective (2-120) can be heated by a heating element. For example, such heating elements maintain the water immersion objective (2-120) at a temperature sufficient to avoid causing temperature changes in the sample contained in the sample plate (2-021).

[0164] 3.1.3. Optomechanical Design and Assembly

[0165] As disclosed herein with respect to specific embodiments, the scanning element (2-022) may be disposed within the optical assembly (2-001) to selectively scan the collimated beam. As can be further observed based on the description provided herein, the scanning element (2-022) may be positioned before or upstream of any additional optical elements that the beam may encounter, i.e., before any additional operations or manipulations are performed on the beam.

[0166] For example, based on certain non-limiting examples described above, in particular embodiments, a scanned collimated beam selectively scanned by the scanning element (2-022) may subsequently be provided to an optical shaping element (2-025) and / or an optical repeater (2-045) for additional optical shaping, guiding, manipulating, and / or other optical operations. As also discussed above, some example optical operations may include (and are not limited to) shaping the beam into a linear and elongated cross-section; homogenizing the beam in a particular direction, such as a longer dimension of the linear cross-section; collimating or converging the beam after another optical operation; guiding, routing, or focusing the beam at one or more specific optical planes of interest.

[0167] In some embodiments, specific optomechanical components may be employed, such as Figures 5A to 5E The optomechanical components referenced and illustrated herein are used to enable rapid and accurate alignment (and realignment, such as for maintenance) and / or adjustment of the optical elements or components disclosed herein, and utilize the optomechanical designs and methods employed therein.

[0168] As a non-limiting example, certain embodiments of this disclosure involve scanning or traversing a collimated beam along the vertex (2-028) of a Powell lens acting as an optical shaping element (2-025), which can produce a beam that (a) remains collimated (e.g., as) in a first plane containing the vertex (2-028) and the optical axis of the scanning lens (2-050) as it exits from the Powell lens acting as the optical shaping element (2-025). Figure 4B (as shown in the diagram), and (b) diverging in a plane orthogonal to the first plane as it leaves the Powell lens, which acts as an optical shaping element (2-025) (as shown in the diagram). Figure 4A (as shown in the diagram), and can subsequently be collimated by suitable optical elements or components (such as a scanning lens (2-050)) to form a collimated linear beam.

[0169] In certain embodiments, such as Figures 5A to 5E As shown, the optical component (2-001) may include an optomechanical sub-component (3-005). In some non-limiting embodiments, the optomechanical sub-component (3-005) may include a scanning component (3-010), an adjustable support mechanism (3-020), and / or an adjustable lens barrel (3-200).

[0170] In a particular embodiment, the scanning assembly (3-010) may include a collimator (2-020), an XY stage (3-008), a galvanometer (2-022) that can be selectively scanned or rotated about its pivot (2-024), and / or an actuator (3-015) for controlling the galvanometer (2-022).

[0171] In a particular embodiment, the adjustable support mechanism (3-020) may include a support rail (3-025), a rotary aligner (3-030), a locking mechanism (3-032) for the rotary aligner, a linear translator (3-035) and / or a locking mechanism (3-038) for the linear translator.

[0172] In a particular embodiment, one or more optical elements or components may be adjustablely supported in, adjustedly supported by, and / or adjustablely supported within the adjustable support mechanism (3-020). As a non-limiting example, a Powell lens acting as an optical shaping element (2-025) may be adjustablely supported within and by the adjustable support mechanism (3-020), such as... Figure 5C As shown in the image.

[0173] A linear translation device (3-035) can be configured to allow lateral adjustment relative to the optical axis of the component and / or a suitable beam. In a particular embodiment, the linear translation device (3-035) can be configured to slide, translate, and / or otherwise suitably move relative to the optical axis of the component. As a non-limiting example, the linear translation device (3-035) can be used to laterally align the apex (2-028) of a Powell lens that acts as an optical shaping element (2-025) with a received beam (such as a collimated beam (2-015) received from a galvanometer (2-022)).

[0174] A rotary aligner (3-030) may be rotatably mounted on an adjustable support mechanism (3-020) so that one or more suitable optical elements or components supported therein may be rotated relative to it. As an illustrative and non-limiting example, the rotary aligner (3-030) may be used to rotatably align the vertex (2-028) of a Powell lens that acts as an optical shaping element (2-025) with the scanning direction of a received beam, such as aligning the scanning direction of a collimated beam (2-015) received from a galvanometer (2-022) with the extent or length of the vertex (2-028).

[0175] Individually or additionally, in certain embodiments disclosed herein, an adjustable support mechanism (3-020) can be used to quickly and accurately align the scanning direction and plane of the collimated beam (2-015) received from the galvanometer (2-022) with the apex (2-028) of the Powell lens, which acts as an optical shaping element (2-025), such that: (a) a uniform and linear beam (e.g., 2-140) in the sample plane (2-130) is properly aligned with the sample plane (2-130) and / or the field of view (FOV), and / or (b) when scanning is performed by a scanning element such as the galvanometer (2-022) , a selectively activated linear photosensitive segment of the detector device (which will be further discussed in the image acquisition section below) is correspondingly aligned with the uniform and linear beam in the sample plane (2-130).

[0176] In a particular embodiment, the adjustable lens tube (3-200) can be used to support and / or quickly and accurately align elements or components within the optical repeater (2-045). As a non-limiting example, the adjustable lens tube (3-200) can support and / or provide one or more adjustable mechanisms for the scanning lens (2-050) and the tube lens (2-090).

[0177] It should be understood that although this disclosure describes particular devices, mechanisms, components and / or methods as non-limiting examples of implementing the aspects disclosed herein, this disclosure contemplates providing any suitable devices, mechanisms, components and / or methods for implementing any such aspects.

[0178] This is publicly available. Figures 5A to 5E A middle region, a lower region, and an upper region of a sample plane are provided according to a specific embodiment.

[0179] Figure 6A Cross-sectional features of an example beam for OLS and alternative methods according to a specific embodiment are shown. In a specific embodiment of the OLS system of this disclosure, the cross-sectional shape of the linear beam appearance of the OLS system of this disclosure is depicted by (4-100). By way of example and not limitation, in some embodiments, (4-100) may correspond to the cross-sectional shape (2-030) previously described herein. In a specific embodiment, a Powell lens acting as an optical shaping element (2-025) may be used to generate the cross-sectional shape (4-100). In other embodiments, such as using other shaping elements (2-025), (4-100) may differ from... Figure 6A The example shown is illustrated in Figure 4-110, which illustrates the intensity profile of the OLS beam corresponding to the longer dimension (4-100) along its cross-sectional shape. In certain embodiments, specific features and / or extents of the OLS beam cross-section may be cropped, filtered, or otherwise shaped to retain a sub-portion (4-150) of the OLS beam thereafter. Figure 6A As shown, the sub-section (4-150) depicted in the non-limiting example can provide uniform intensity along its longer dimension. Individually or additionally, as shown, the sub-section (4-150) can be formed from the total cross-sectional area of ​​the complete beam (4-100) and / or a significant portion of the beam energy. Therefore, the sub-section (4-150) of a particular OLS embodiment of this disclosure can provide a combination of uniform intensity of the OLS beam and / or high overall illumination efficiency. As a further result, more efficient and / or compact illumination modules can be provided, along with lower heat generation and less need for cooling and stabilization.

[0180] In contrast to the embodiments (4-150) and OLS disclosed herein, alternative methods may employ beam generation and / or shaping techniques that can provide beams with relatively highly non-uniform cross-sectional shapes and / or intensities, as shown in (4-200). A light intensity profile corresponding to the beam cross-sectional appearance of (4-200) is shown in (4-210).

[0181] As an example, in such alternative techniques, the need for more uniform beam intensity across a longer dimension of the cross-section can be met by cropping, windowing, filtering, or otherwise extracting a sub-section (4-250) of the overall incident beam (4-200) (such as its narrow central region). Therefore, such a sub-section (4-250) may suffer from low overall illumination efficiency due to at least poor utilization of the overall incident beam (4-200). Consequently, larger and heavier illumination modules and components, as well as support modules for cooling and thermal stabilization of the generated heat, may be required.

[0182] As a further example, in such alternative techniques, the need for higher overall optical efficiency may prompt the cropping, windowing, filtering, or other methods to extract a sub-section (4-260) of the overall beam (4-200), with a cross-section (4-260) wider than that of (4-250). Such methods may have the disadvantage of strong beam non-uniformity along at least the longer dimension of the cross-section.

[0183] Therefore, as Figure 6A As shown in the non-limiting examples, certain aspects of the OLS embodiments disclosed herein provide means for simultaneously generating uniform and efficient sample illumination.

[0184] 3.1.4. Image Acquisition

[0185] In some non-limiting embodiments of the image acquisition system disclosed herein, the objective lens (2-120) may also be used to focus fluorescence emitted by the sample (2-145) in response to illumination provided by the tilting beam (2-125). In some non-limiting embodiments, the fluorescence emission (2-145) focused by the objective lens may pass through an emission filter ((2-150) and / or (2-160)), for example, a bandpass emission filter spectrally matched to the observed fluorophore and mounted in a high-speed filter wheel (Finger Lakes Instruments), and be collected by the detector device (2-165). In some non-limiting embodiments, the fluorescence emission focused by the objective lens may be directed to an optical tube or repeater before being collected by the detector device (2-165). For example, but not as a limitation, such an optical tube or repeater may include one or more lenses (e.g., tube lens 2-155), and one or more additional optical elements, for example, elements configured to suppress additional scattered light before being collected by the detector device (2-165). In some non-limiting embodiments, the fluorescence emission focused by the objective lens is directed through another dichroic mirror to split the emission across multiple regions of the detector (2-165). In some non-limiting embodiments, the fluorescence emission focused by the objective lens may be directed through another dichroic mirror to split the emission across multiple detectors (2-165).

[0186] Figure 6BA rolling shutter operation according to a particular embodiment is illustrated, depicting an example of the relative timing and synchronization of sensor exposure and sample illumination scanning. In some non-limiting embodiments of the image acquisition system of this disclosure, the detector device can be configured to synchronize the detection and / or activation of the photosensitive segment with the translation of the tilted beam (2-125) across the sample plane (2-130). Such synchronization in Figure 6B The diagram is schematically depicted. For example, but not as a limitation, the detector device can be a CMOS camera, such as a back-illuminated CMOS camera (Hamamatsu Fusion BT). In a particular embodiment, selective activation of the photosensitive segment synchronized with the scanning beam (2-125) across the sample plane (2-130) can provide a confocal effect that selectively filters out incident light from the defocus plane.

[0187] As an example, not a limitation, Figure 6B A detector device (2-165) is shown, such as a CMOS sensor including sensor elements (such as pixels). One or more columns of pixels (4-310) can be activated at certain time intervals and provide imaging light from an illuminating sample plane during that given time period. The activated pixel columns (4-310) of the sensor can be swept or traversed in a timely manner, synchronized with the scanning or translation of the beam at the sample plane, which can in turn be achieved by synchronously scanning scanning elements (2-022) (such as galvanometers).

[0188] As a non-limiting example, see [reference] Figure 6B In a particular embodiment, all pixels in the first column (such as (4-310-1)) can be simultaneously exposed to imaging light received from the illumination portion of the sample plane, starting at time point (4-320-1) and ending at time point (4-330-1), i.e., during the specific column exposure time interval (4-350-1). During the aforementioned exposure time interval (4-350-1) of column (4-310-1), other columns (4-310-2) to (4-310-n) of pixels can be deactivated, thereby providing a confocal effect that eliminates background light from other areas of the sample plane that may not be intended for imaging during the exposure time interval (4-350-1).

[0189] At another time point (4-320-2), which in a particular embodiment may occur before the completion time point (4-330-1) of the exposure of the previous column, all pixels of the second column (such as (4-310-2)) may be simultaneously exposed to imaging light received from the sample plane, wherein the illuminated portion of the sample plane may be swept or scanned accordingly by the scanning element, synchronized with the column activation sweep rate of the sensor. Other columns of pixels may be deactivated during the exposure time interval of the second column (4-310-2), including one or more previously activated columns after their respective exposure time intervals have completed. This process may continue until the last column of pixels of the sensor (such as (4-310-n)) has completed the process. Thus, a total imaging time interval for imaging the complete FOV can be defined, such as Figure 6B As shown in (4-360).

[0190] In some embodiments, the image acquisition system of this disclosure is configured to allow analysis of one or more alignment parameters. For example, Figure 7A Results obtained in relation to determining the tilt angle of an exemplary tilted sheet are shown. In some embodiments of such analyses, for each row of the recorded z-stack, Gaussian fitting model statistics provide a non-limiting example of the position of the exemplary tilted sheet. The maximum peak amplitude of the sheet (in pixel values) provides an exemplary measure of the sheet position perpendicular to the tilt axis at a given location along the z-stack axis. The minimum sheet width (in pixel values) provides an exemplary measure of the sheet waist along the beam propagation. Linear fitting model statistics provide a non-limiting example of the tilt angle of the exemplary tilted sheet.

[0191] Figure 7B and Figure 7C Non-limiting examples of results obtained in relation to scanning optimization of exemplary scanning tilted light plates are provided. For example, but not as a limitation, Figure 7B The illustration shows spot detection on an exemplary scanning tilted film containing a stationary fluorescent dot source. Spot detection statistics, including but not limited to spot count, top transition, and bottom transition, provide exemplary measures of spatial alignment of the exemplary scanning tilted film and camera-detected FOV. In some embodiments, spot count, top transition, and bottom transition also provide exemplary measures of temporal synchronization of the exemplary scanning tilted film and camera-detected FOV. In some embodiments, one or more of a flatness measure and a slope measure provide exemplary measures of FOV uniformity. Additionally, cost measures may include a comprehensive score based on the above-described scanning optimization measures. Similarly, Figure 7CResults of a non-limiting example of an iterative scan optimization analysis process are shown. In some embodiments, camera-triggered offsets and galvanometer amplitudes constitute non-limiting parameters that can be analyzed. Similarly, cost metrics can provide an exemplary measure of scan optimization with varying scan parameters in such an iterative environment. In some embodiments, Gaussian fitting model statistics can provide a measure of the scan optimization analysis process.

[0192] Figure 7D Non-limiting examples of the types of results that can be obtained during speckle detection analysis are shown. In some embodiments, speckle detection statistics can facilitate scan optimization. For example, but not as a limitation, attributes such as mean SNR, SNR skewness, SNR CV, SNR Frac. Pix, and Detect. Frac. Pix. can be used in conjunction with speckle detection of an exemplary scanned tilted sheet and indicate the uniformity of the camera detection FOV. Additional features, such as, but not limited to, PSF major axis FWHM, PSF minor axis FWHM, and PSF symmetry, can be performed as metrics for speckle detection of an exemplary scanned tilted sheet and facilitate the analysis of optical aberrations.

[0193] Figure 7E Non-limiting examples of the types of results that can be obtained when aligning an exemplary scanning tilted beam are shown. For example, but not as a limitation, linewidth statistics can provide information about, for example, the width of an exemplary beam, including the width measured along a direction perpendicular to the longer dimension of the beam (the Y-width in these illustrations). Furthermore, the median peak amplitude of the beam (represented in pixel values) can provide an exemplary measure of beam intensity. Similarly, positional measurements ( Figure 7E The Y-position in the figure provides a numerical reference (Y-position) for the beam position corresponding to the corresponding region for each example. In some embodiments, the tilt angle can provide a horizontal reference (i.e., an alignment measure relative to the X-direction) for the FOV of each exemplary beam relative to the sample plane. In some embodiments, the SAG value can provide a numerical measure of the beam's sag or curvature along a longer dimension of the beam. In some embodiments, the CV(x) value can provide an exemplary measure of the variability of pixel values ​​or intensity along a longer dimension of the exemplary beam, where CV(x) refers to the coefficient of variation along the longer dimension (X-direction) of the beam and is defined as the standard deviation of pixel values ​​obtained along the longer dimension (X-direction) of the beam divided by the average pixel value of the beam. The non-limiting maximum pixel value corresponding to the example shown in this figure is 65536 (i.e., 2^16), far exceeding the peak median pixel value of approximately 30000.

[0194] Although specific configurations, sequences, and / or orientations for illumination, exposure, sweeping, scanning, and / or sensor activation and deactivation may be provided in this disclosure for illustrative purposes, this disclosure considers any suitable configuration, sequence, and / or orientation to provide the functional and optical objectives described herein.

[0195] In certain embodiments, in addition to or instead of the rolling shutter operation described above, one or more physical slits, pinholes, or equivalent optical elements or components may be used to provide confocality.

[0196] In some embodiments of the image acquisition system disclosed herein, the CMOS camera may operate such that, for each field of view, a series of SMT frames are collected. For example, but not limited to, 1-20,000 SMT frames, 1-15,000 SMT frames, 1-10,000 SMT frames, 1-5,000 SMT frames, 1-1,000 SMT frames, 2-500 SMT frames, 5-250 SMT frames, 10-200 SMT frames, 100-200 SMT frames, or 200 SMT frames are collected per field of view. In some embodiments, the CMOS camera may be configured to operate at a frame rate of 0.5 to 1000 Hz, or in some embodiments, at a frame rate of 100 Hz. For example, but not limited to, some cellular SMT implementations may operate at 100 Hz.

[0197] In some non-limiting embodiments of the image acquisition system disclosed herein, the detector device is configured to transmit a signal with each frame to trigger other elements of the imaging system. For example, but not as a limitation, the detector device may trigger illumination from a light source (2-005) to collect fluorescence emission associated with a stroboscopic laser pulse. For example, but not as a limitation, such fluorescence emission collection may be associated with 10 to 100 msec frames and 2 msec stroboscopic laser pulses.

[0198] In some implementations, the imaging acquisition system can be configured to acquire a predetermined image size per frame, referred to herein as a region of interest (ROI). In some embodiments, the ROI will vary depending on the frame rate used. For example, at 100 FPS, 2304 x 1728 pixels would define the ROI, which corresponds to 248.832 x 186.624 micrometers in the sample plane. In contrast, at 200 FPS, 2304 x 768 pixels would define the ROI, which corresponds to 248.832 x 82.944 micrometers in the sample plane.

[0199] In some implementations, the imaging acquisition system can be configured to perform a predetermined sweep rate at a predetermined frame rate. For example, but not by limitation, at 100 FPS: the sweep rate could be 186.624 μm / 9 ms, which is equivalent to 20.8 μm / ms, or 2.08 cm / s. In contrast, at 200 FPS, the sweep rate could be 82.94 μm / 4 ms, which is equivalent to 20.7 μm / ms, or 2.07 cm / s.

[0200] In some embodiments, the detector device can be used to collect fluorescence emission at multiple wavelengths. For example, but not as a limitation, fluorescence emission from additional fluorophores can be collected for the same field of view at the same or different frame rates to provide downstream registration of the SMT trajectory with other cellular components, such as the cell nucleus. Additional channels of the detector device can be used as needed to expand the number of fluorescence emissions captured simultaneously for the same field of view to provide downstream registration of the SMT trajectory with other cellular components, such as the cell nucleus.

[0201] 4. Exemplary Single-Molecule Tracking Software

[0202] Figure 8 An exemplary sample processing system of this disclosure is shown. Figure 9 An example system 600 for measuring molecular motion (e.g., motion in living cells) is shown. Experiment 602 can be performed to collect large amounts of data from multiple living cells (e.g., using imaging system 624 to identify compound 626 and / or target 622). Experiment 602 may include applying various identifiers to molecules of interest, such as labels that can subsequently be fluorescently or otherwise detected (e.g., using lasers or other light sources). Biological samples forming part of such experiment 602 may be organized into a plate 604 having multiple wells 606. Each well 606 may have one or more associated fields of view (FOVs) 610. FOV 610 may be within a single well 606 or at a location corresponding to that single well. A series of images can be generated for FOV 610 to produce one or more films 612, which may include SMT films as well as non-SMT films. SMT films can be used to trace the path of a single labeled molecule, such as a protein, thereby generating multiple trajectories. Each trajectory may consist of multiple spots 614, which include the spatiotemporal coordinates of the labeled molecule at a specific time (e.g., ...). Figure 10 (As described in further detail below). Separately to, and in some instances in parallel with, tracking, the film 612 can be used to identify molecules to generate a mask 618 by using machine learning and / or computer vision-based image segmentation. The mask 618 is a spatial region within the FOV 610 generated by segmentation. Each mask 618 can belong to a mask category, which is... Figure 10A more detailed description is provided below.

[0203] Data associated with two channels (e.g., a tracking channel and a segmentation / masking channel) can be combined to generate multiple metrics 620 associated with various aspects of the sample. In other words, trajectories 616 (e.g., trajectory data) can be combined with image segmentation data processed by machine learning and further analyzed using statistical / machine learning methods. The processing of the combined data can be used to generate metrics 620, such as hit scores associated with compounds and / or targets within the biological sample, which can be stored in a database structure, such as... Figure 12 Further details are provided below.

[0204] Figure 10 A data stream is illustrated using an example system 700 of a high-throughput single-molecule imaging platform for measuring protein motility in living cells. Experimental specifications 704 defining experiment 602 can be provided as data input via one or more clients 702. For example, each experiment 602 can be collected using accompanying staining agents (e.g., Hoechst or PotomacRed) for downstream analyses including segmentation 618. Experimental specifications 704 can define various parameters for experiment 602, such as staining agents, dyes, compounds, treatments, etc. As previously described in... Figure 9 As described, imaging system 706 (e.g., imaging system 624) can capture image sequences that generate one or more SMT films 711 and / or non-SMT films or segmented films 708 (e.g., film 612) characterizing molecular motion. SMT film 711 can characterize the motion of a single fluorescent molecule and / or contain an image of a single fluorescent molecule. Segmented film 708 can include image sequences characterizing the motion of labeled molecules and / or their components. It should be understood that Hoechst staining is only one technique that can be used to label molecules, and different and / or multiple labeling techniques, such as Potomac Red, can be utilized depending on the desired configuration. For example, MitoTracker™ Deep Red can be used to label mitochondria, concanavalin A-dye conjugates can be used to label the endoplasmic reticulum, SYTO 14 can be used to label the nucleolus, phalloidin can be used to label actin, etc.

[0205] The SMT video 711 can be analyzed to perform operations related to molecular tracing 710, which may include detection 712, subpixel localization 713, and linking 714 to identify molecular trajectories 715 across various images within the SMT video 711. More specifically, during detection 712, one or more spots within the SMT video 711 can be detected or recovered. Each spot may be equipped with spatiotemporal coordinates. These spatiotemporal coordinates can be estimated using subpixel localization technology 713. Linking 714 can be performed on the spots to ultimately identify the trajectory 715.

[0206] As used in this paper, a link is a potential association between two blobs. Each link is guided to begin at one blob and end at another. A “correct link” joins two blobs generated by the same emitter in different frames; otherwise, the link is “incorrect.” One goal of the linking algorithm is to estimate which links are correct. This is presented in the format... Links are mentioned. This is understood as: a link α that starts at spot i and ends at spot j. A link satisfies at least three of the following constraints: (a) the link is forward in time, (b) a link cannot join two spots that are more than a certain limit (referred to as the “search radius” in this paper), and (c) a link cannot join two spots that are more than a certain limit (referred to as the “gap limit” in this paper) in time. A spot-link graph is a graph of spots and links in an SMT movie 711. Spots are the vertices of the graph, and links are the edges of the graph. Since links are forward in time, the spot-link graph is a directed acyclic graph. A match is a subset of links in a spot-link graph such that no two links in the subset start or end at the same spot. In this paper, a trajectory 715 is used to refer to a sequence of consecutive (end-to-end) links in the same match. Multiple trajectories can be used to determine the dynamic metric 730. Such parameters can include spot properties characterizing spot motion. Such parameters can include one or more of the velocity, diffusion coefficient, or anomaly parameter for each spot. The dynamic parameter of spot i is referred to as θ in this paper. i The set of dynamic parameters for all blobs in the blob-link diagram is referred to in this paper as... .

[0207] Separate from and in parallel with the processing of SMT film 711 in some variations, segmentation film 708 may undergo segmentation, which generates one or more masks 720. Masks can be of various categories, including but not limited to nucleus, cytoplasm, and / or irrelevant masks, which in... Figure 11 Further description follows. An instance mask is a single segmented object (e.g., a cell, a nucleus, a mitochondrion). FOV 610 can contain any number of instance masks for a mask category. A semantic mask is the union of all instance masks corresponding to a mask category of a FOV (e.g., all cells, all nuclei, or all mitochondria of a FOV). Irrelevant masks can contain portions of non-SMT film 708 that are excluded from any downstream data analysis. For example, these irrelevant masks may correspond to out-of-focus portions of non-SMT film 708 or portions containing autofluorescent cell debris that prevents accurate tracking. During segmentation, molecules within segmented film 708 can be assigned to one or more masks. Image metrics 740, such as cell health, focus quality, etc., can be evaluated from mask molecules.

[0208] Experimental information, such as kinetic metric 730, image metric 740, and any data derived from any of these metrics (e.g., segmentation information), can be provided to a data repository 770 for storage. This data repository 770 can store, for example, any results of experiment 602, such as kinetic metric 730, image metric 740, and / or any data derived from any of these metrics. The data repository may include a dedicated server with local persistence and / or local access or access via the cloud. The data repository 770 may also store metadata associated with it and / or metadata associated with experiment specification 704. Experimental information (e.g., results and metadata of historical experiments) can be provided to the data repository 770 via a repository application programming interface (API) 750. The repository API 750 can also interface with a web-based graphical user interface front-end 760, which provides this information for display on a client 702.

[0209] In some variations, segmentation information can be used to identify subcellular compartments, such as the nucleus, nucleolus, and cytoplasm. Segmentation information can also be used to distinguish one cell from another. Segmentation information can be stored in a specific format (e.g., multiple image file formats such as TIFF).

[0210] Example kinetics metric 730 can also include a state array. A state array is a framework for learning interpretable kinetic models from SMT trajectories and can be used to gain additional insights into target protein motion and where that motion occurs within the cell. In some variations, segmentation information can be used to generate / fill the state array. The output of the state array can be returned at the subcellular compartment level, allowing scientists to distinguish the kinetics within different subcellular compartments. Furthermore, the state array can be computed on each individual subcellular compartment (e.g., each nucleus).

[0211] To facilitate data access by applications (including, but not limited to, state arrays), processed SMT data can be stored in a format that allows: (a) representations of processed trajectories and associated attributes, such as the SNR and blob shape characteristics of each SMT video; (b) representations of mask objects, including mask categories (e.g., subcellular organelles associated with each mask object); (c) associations between trajectories and mask objects (e.g., the nuclei observed in each trajectory); and (d) associations of all SMT videos with metadata related to the original experiment, such as compound treatment, acquisition time, and imaging system name. Formats (a) and (c) can be Protocol Buffer patterns, which define the storage format for trajectories and associated mask objects. Format (b) can be a specialized image file format that includes the mask object to which each pixel in the FOV belongs. Format (d) can be a PostgreSQL database recording all captured experiments / videos. As a client of processed SMT data, state arrays can utilize these data patterns to report dynamic characteristics of trajectories for each mask category or each mask object.

[0212] Figure 11 Multiple images 800 illustrate the differences between mask categories and instance or semantic masks. As previously discussed, non-SMT or segmented videos can be assigned to multiple categories. Such categories may include nuclei (e.g., category A), cytoplasm (e.g., category B), and / or irrelevant masks (e.g., category C). Unique individual masks can be applied to biological samples. For example, image 810 belongs to a unique individual instance mask applied to nuclei (e.g., category A). Image 812 belongs to a unique individual instance mask applied to cytoplasm (e.g., category B). Image 820 shows multiple instance masks applied to one or more nuclei, where each color represents a different unique individual instance mask. Image 822 shows multiple masks applied to one or more cytoplasms, where each color represents a different unique individual instance mask. Image 830 shows a semantic mask applied to one or more nuclei, which is the union of all instance masks. Image 832 shows a semantic mask applied to one or more cytoplasms.

[0213] Figure 12 An example computer implementation environment 900 is shown, in which the imaging system 910 can interact with the computing architecture to execute the various algorithms described herein. Figure 12As shown, the imaging system 910 may interface with one or more clients 950 (e.g., via client 702 of a web application with a graphical user interface). One or more clients 950 may interface with one or more servers 920 accessible via network 930. One or more clients 950 may host frame captures of images (e.g., video 612) captured from a camera. Those images may be temporarily stored on one or more clients 950 and periodically transmitted via network 930 to one or more servers 920 for remote storage. One or more servers 920 may also contain or access one or more data storage units 940 for storing data collected and / or extracted from a sample by the imaging system 910. In some variations, network 930 may include or interface with one or more network storage arrays 960 for storing data such as captured images (e.g., video 612).

[0214] Figure 13 Figure 1000 illustrates a sample computing device architecture for implementing the various aspects described herein. In some variations, the sample computing device architecture may be the architecture of client 950 and / or server 920, and some components described in relation to Figure 1000 may be optional for client 950 and / or server 920. Bus 1004 may serve as an information highway for interconnecting other illustrated components of the hardware. Processing system 1008, labeled CPU (Central Processing Unit) (e.g., one or more computer processors / data processors at a given computer or multiple computers), may perform computational and logical operations required to execute a program. Optionally or additionally, processing system 1012, labeled GPU (Graphics Processing Unit) (e.g., one or more computer processors / data processors at a given computer or multiple computers), may perform computational and logical operations required to execute a program. Non-transitory processor-readable storage media, such as read-only memory (ROM) 1016 and random access memory (RAM) 1020, may communicate with processing system 1008 and / or processing system 1012 and may include one or more programming instructions for the operations specified herein. Optionally, program instructions may be stored on a non-transitory computer-readable storage medium, such as a disk, optical disk, recordable storage device, flash memory, solid-state drive, or other physical storage medium.

[0215] In one example, disk controller 1048 may interface to system bus 1004 using one or more optional removable storage devices 1056 or local storage devices 1052. Removable storage device 1056 may be an external or internal disk drive, a solid-state drive, or an external hard disk drive. Local storage device 1052 may be an internal hard disk drive and / or memory. As previously described, these various examples of removable storage device 1056, local storage device 1052, and disk controller 1048 are optional devices. System bus 1004 may also include at least one communication interface 1024 to allow communication with external devices physically connected to the computing system or externally available via wired or wireless networks, such as cloud storage devices and remote services. In some cases, at least one communication interface 1024 includes or otherwise incorporates a network interface.

[0216] In some variations, such as for client 950, to provide interaction with the user, the subject matter described herein can be implemented on a computing device having a display device 1044 (e.g., an LCD (liquid crystal display) or LED (light-emitting diode) monitor) to display information obtained from bus 1004 to the user via display interface 1040, and an input device 1032, such as a keyboard and / or pointing device (e.g., a mouse or trackball) and / or touchscreen, through which the user can provide input to the computer. Other types of input devices 1032 can also be used to provide interaction with the user; for example, feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback via microphone 1036, or tactile feedback); and input from the user can be received in any form, including acoustic, voice, or tactile input. Input device 1032 and microphone 1036 can be coupled to bus 1004 via input device interface 1028 and information can be transmitted via the bus. As an example, input device 1032 can be an imaging system 910 configured to have the capability to capture image sequences as described herein. Frame capturer 1058 can capture or extract individual frames from analog or digital data encapsulating an image sequence obtained from bus 1004. Frame capturer 1058 may include memory capable of storing one or more frames. Frame capturer 1058 may also provide one or more frames to bus 1004 for further storage, such as on local storage device 1052 and / or removable storage device 1056. Other computing devices, such as dedicated servers, may omit the integration. Figure 13 One or more of the components being described.

[0217] One or more aspects or features of the subject matter described herein can be implemented in digital electronic circuit systems, integrated circuit systems, application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), computer hardware, firmware, software, and / or combinations thereof. These different aspects or features can be implemented in one or more computer programs that can be executed and / or interpreted on a programmable system comprising at least one programmable processor, which may be coupled for special or general purposes to receive data and instructions from a storage system, at least one input device, and at least one output device, and to transfer data and instructions to the storage system, at least one input device, and at least one output device. The programmable system or computing system may include clients and servers. Clients and servers are typically geographically separated and typically interact via a communication network. The client-server relationship is generated by means of computer programs running on respective computers and having a client-server relationship with each other.

[0218] These computer programs (also referred to as programs, software, software applications, applications, components, or code) contain machine instructions for a programmable processor and can be implemented using high-level programming languages, object-oriented programming languages, functional programming languages, logic programming languages, and / or assembly / machine languages. As used herein, the term "machine-readable medium" refers to any computer program product, device, and / or apparatus for providing machine instructions and / or data to a programmable processor, such as a disk, optical disk, memory, and programmable logic device (PLD), containing a machine-readable medium that receives machine instructions as a machine-readable signal. The term "machine-readable signal" refers to any signal used to provide machine instructions and / or data to a programmable processor. Machine-readable media can store such machine instructions non-transitory, such as like non-transitory solid-state memory or magnetic hard disk drives or any equivalent storage medium. Machine-readable media can alternatively or additionally store such machine instructions in a transient manner, such as like a processor cache or other random access memory associated with one or more physical processor cores.

[0219] 5. Example

[0220] Exemplary solution for manufacturing a reference sample plate

[0221] 5.1 Exemplary Compositions

[0222] Qdot-605 stock solution (QDS), 20 μL of Qdot605 probe in 600 μL distilled water and 3 μL TWEEN 20™ (ThermoFisher Scientific).

[0223] Qdot-605 dilution solution (QDD). 1 μL of QDS in 5 mL of distilled water.

[0224] QSY™ 9, 0.25% w / v, in distilled water.

[0225] Rhodamine-6G (R6G), 10 mM, in DMSO

[0226] Quenched R6G (R6GQ) was mixed with 25 μL of QSY™ 9.

[0227] N,N'-Methylenebis(acrylamide) (MBA), 1.53 mg / ml (0.153% w / v), in Tris-HCl (pH 7.5)

[0228] Acrylamide, 0.306 g / ml (30.6% w / v), in Tris-HCl (pH 7.5)

[0229] Tetramethylethylenediamine (TEMED), pure

[0230] Polyacrylamide stock solution (PAA). Just before use, mix 1 mL of acrylamide solution with 0.09 mL of MBA and 10 μL of TEMED.

[0231] Ammonium persulfate (APS), 12.5 mg / ml, in Tris-HCl (pH 7.5)

[0232] Low melting point agarose, 20 mg / ml, in FluoroBrite™ DMEM.

[0233] 384-hole plate.

[0234] Bath-type ultrasonic instrument.

[0235] 70℃ oscillating heating block.

[0236] 5.2 Exemplary Method

[0237] All solutions should be sonicated for 30 minutes before use.

[0238] 5.2.1 Polyacrylamide hydrogel lamination

[0239] Mix PAA (300 μL) with QDS (1 μL), sonicate for 30 min, then mix with APS (50 μL). Aliquot the 50 μL mixture into appropriate wells of a 384-well plate (e.g., B2, B23, G9-G16, I9-I16, O2, and O23; see [link to article]). Figure 2This allows the mixture to spread evenly and contact the inner corner of each hole.

[0240] 5.2.2 Low Melting Point Agarose (LMA) Hydrogel Lamination

[0241] LMA (300 μL) and QDS (1 μL) were mixed in an Eppendorf tube and sonicated for 30 minutes. The tube was then heated at 70°C and 800 rpm for 3 minutes. 50 μL of the heated mixture was aliquoted into appropriate wells of a 384-well plate (e.g., C3, C22, H9-H16, J9-J16, N3, and N22; see [link to article]). Figure 2 This allows the mixture to spread evenly and contact the inner corner of each hole.

[0242] 5.2.3 R6G and R6GQ PCB Lamination

[0243] Pipe 50 μL of R6G or R6GQ into the appropriate wells of a 384-well plate (e.g., G8 for R6G and H8 for R6GQ, see...). Figure 2 ).

[0244] 5.2.4 Qdot-605 dilution solution (QDD) plate preparation

[0245] Pipe 50 μL of QDD into the appropriate wells of the 384-well plate (e.g., E6-E19 and F6-F19, see...). Figure 2 ).

[0246] Store the 384-well plate uncovered overnight in a cleanroom environment at room temperature, then seal until used.

Claims

1. A composition comprising a plurality of individually addressable reference samples, wherein the reference samples are selected from: A. A reference sample containing multiple spatially stationary optical point sources; B. A reference sample containing multiple diffusible optical point sources; C. A reference sample containing a uniform optical volume; and D. A reference sample containing one or more resolution test patterns.

2. The composition of claim 1, wherein the reference sample is contained in an individually addressable sample chamber of the sample container.

3. The composition according to claim 2, wherein each sample chamber comprises: A. A bottom surface made of an optically permeable material; as well as B. A vertical wall formed along the periphery of the bottom surface, in which a cavity is formed by a closed bottom end and an open top end.

4. The composition according to claim 1, wherein the plurality of spatially stationary optical point sources are fixed in the polymer.

5. The composition according to claim 4, wherein the polymer is a hydrogel.

6. The composition according to claim 5, wherein the hydrogel is a polysaccharide hydrogel, polyacrylamide hydrogel, polyacrylic acid hydrogel, polymethyl acrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or a combination thereof.

7. The composition according to claim 6, wherein the hydrogel is a polyacrylamide hydrogel or an agarose hydrogel.

8. The composition according to claim 4, wherein the polymer is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene, polycarbonate, or polyvinyl butyral.

9. The composition of claim 4, wherein the polymer is degassed or homogenized and degassed.

10. The composition of claim 4, wherein the spatially stationary optical point source is immobilized in the polymer by covalent bonding, adsorption, electrostatic bonding, or a combination thereof.

11. The composition of claim 1, wherein the spatially stationary optical point source is fixed within a 3D lattice formed of the polymer.

12. The composition according to any one of claims 1 to 11, wherein the plurality of spatially stationary optical point sources are a plurality of spatially stationary fluorescent point sources.

13. The composition of claim 12, wherein the plurality of spatially stationary fluorescent dot sources comprise quantum dots.

14. The composition of claim 13, wherein the quantum dot has an emission maximum of about 400 nm to about 720 nm.

15. The composition of claim 14, wherein the quantum dot has an emission maximum of about 420 nm to about 480 nm.

16. The composition of claim 15, wherein the quantum dot has an emission maximum of 450 nm.

17. The composition of claim 14, wherein the quantum dot has an emission maximum of about 500 nm to about 550 nm.

18. The composition of claim 17, wherein the quantum dot has an emission maximum of 525 nm.

19. The composition of claim 17, wherein the quantum dot has an emission maximum of 545 nm.

20. The composition of claim 14, wherein the quantum dot has an emission maximum of about 525 nm to about 575 nm.

21. The composition of claim 20, wherein the quantum dot has an emission maximum of 565 nm.

22. The composition of claim 14, wherein the quantum dot has an emission maximum of about 575 nm to about 650 nm.

23. The composition of claim 22, wherein the quantum dot has an emission maximum of 585 nm.

24. The composition of claim 22, wherein the quantum dot has an emission maximum of 605 nm.

25. The composition of claim 22, wherein the quantum dot has an emission maximum of 625 nm.

26. The composition of claim 14, wherein the quantum dot has an emission maximum of about 650 nm to about 720 nm.

27. The composition of claim 26, wherein the quantum dot has an emission maximum of 655 nm.

28. The composition of claim 26, wherein the quantum dot has an emission maximum of 705 nm.

29. The composition of claim 12, wherein the plurality of spatially stationary fluorescent dot sources comprise nanodiamonds.

30. The composition of claim 29, wherein the nanodiamond has an emission maximum of about 400 nm to about 720 nm.

31. The composition of claim 30, wherein the nanodiamond has an emission maximum of about 400 nm to about 500 nm.

32. The composition of claim 31, wherein the nanodiamond has an emission maximum of about 415 nm.

33. The composition of claim 30, wherein the nanodiamond has an emission maximum of about 500 nm to about 550 nm.

34. The composition of claim 33, wherein the nanodiamond has a maximum emission value of 510 nm.

35. The composition of claim 30, wherein the nanodiamond has an emission maximum of about 550 nm to about 600 nm.

36. The composition of claim 35, wherein the nanodiamond has a maximum emission value of 575 nm.

37. The composition of claim 30, wherein the nanodiamond has an emission maximum of about 600 nm to about 700 nm.

38. The composition of claim 37, wherein the nanodiamond has an emission maximum of 638 nm.

39. The composition of claim 1, wherein the plurality of diffusing optical point sources are disposed in the solution.

40. The composition of claim 39, wherein the solution comprises Tris:HCl, DMSO, DMEM, DPBS, or H2O.

41. The composition of claim 39, wherein the solution comprises a polytungstate.

42. The composition of claim 39, wherein the solution comprises polysorbate 20 or (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol).

43. The composition of claim 1, wherein each of the diffusing optical point sources comprises a conjugated compound.

44. The composition of claim 43, wherein the compound is a protein, polyethylene glycol, polysaccharide, oligonucleotide, polyamine or polyamino acid, or derivatives thereof and analogs thereof.

45. The composition according to claim 43, wherein the compound is a protein selected from ovalbumin, serum albumin and avidin.

46. ​​The composition of claim 43, wherein the compound is a polyamino acid selected from polylysine, polyhistidine, polyglutamic acid, polyaspartic acid, derivatives thereof, and analogs thereof.

47. The composition of claim 43, wherein the compound is polyethylene glycol, its derivatives and analogs having a molecular weight of about 200 Da to about 6000 Da.

48. The composition of claim 43, wherein the plurality of diffusing optical point sources are housed in individually addressable sample chambers, and the compound is operatively attached to the surface of the sample chambers.

49. The composition of claim 43, wherein the compound is attached to the surface of the sample chamber and to the diffusing optical point source.

50. The composition of claim 49, wherein the compound is attached at a first end to the bottom surface of the sample chamber and at a second end to the diffusing optical point source.

51. The composition of claim 50, wherein the coupling is performed by covalent bonding, adsorption, electrostatic bonding, hydrophobic bonding, or a combination thereof.

52. The composition of claim 1, wherein the plurality of diffusing optical point sources are disposed in the polymer.

53. The composition of claim 52, wherein the polymer is a hydrogel.

54. The composition of claim 53, wherein the hydrogel is a polysaccharide hydrogel, polyacrylamide hydrogel, polyacrylic acid hydrogel, polymethyl acrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or a combination thereof.

55. The composition according to claim 53, wherein the hydrogel is a polyacrylamide hydrogel or an agarose hydrogel.

56. The composition according to claim 52, wherein the polymer is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene (PTFE), polycarbonate, or polyvinyl butyral.

57. The composition according to any one of claims 39 to 56, wherein the plurality of diffusible optical point sources comprise quantum dots.

58. The composition of claim 57, wherein the quantum dot has an emission maximum of about 400 nm to about 720 nm.

59. The composition of claim 58, wherein the quantum dot has an emission maximum of about 420 nm to about 480 nm.

60. The composition of claim 58, wherein the quantum dot has an emission maximum of 450 nm.

61. The composition of claim 58, wherein the quantum dot has an emission maximum of about 500 nm to about 550 nm.

62. The composition of claim 61, wherein the quantum dot has an emission maximum of 525 nm.

63. The composition of claim 61, wherein the quantum dot has an emission maximum of 545 nm.

64. The composition of claim 58, wherein the quantum dot has an emission maximum of about 525 nm to about 575 nm.

65. The composition of claim 64, wherein the quantum dot has an emission maximum of 565 nm.

66. The composition of claim 58, wherein the quantum dot has an emission maximum of about 575 nm to about 650 nm.

67. The composition of claim 66, wherein the quantum dot has an emission maximum of 585 nm.

68. The composition of claim 66, wherein the quantum dot has an emission maximum of 605 nm.

69. The composition of claim 66, wherein the quantum dot has an emission maximum of 625 nm.

70. The composition of claim 58, wherein the quantum dot has an emission maximum of about 650 nm to about 720 nm.

71. The composition of claim 70, wherein the quantum dot has an emission maximum of 655 nm.

72. The composition of claim 70, wherein the quantum dot has an emission maximum of 705 nm.

73. The composition according to any one of claims 39 to 56, wherein the plurality of diffusible optical point sources comprise nanodiamond.

74. The composition of claim 73, wherein the nanodiamond has an emission maximum of about 400 nm to about 720 nm.

75. The composition of claim 74, wherein the nanodiamond has an emission maximum of about 400 nm to about 500 nm.

76. The composition of claim 75, wherein the nanodiamond has an emission maximum of about 415 nm.

77. The composition of claim 74, wherein the nanodiamond has an emission maximum of about 500 nm to about 550 nm.

78. The composition of claim 77, wherein the nanodiamond has a maximum emission value of 510 nm.

79. The composition of claim 74, wherein the nanodiamond has an emission maximum of about 550 nm to about 600 nm.

80. The composition of claim 79, wherein the nanodiamond has an emission maximum of 575 nm.

81. The composition of claim 74, wherein the nanodiamond has an emission maximum of about 600 nm to about 700 nm.

82. The composition of claim 81, wherein the nanodiamond has an emission maximum of 638 nm.

83. The composition according to claim 1, wherein the uniform optical volume is disposed within the matrix.

84. The composition of claim 83, wherein the matrix is ​​a solution or a polymer.

85. The composition of claim 84, wherein the solution comprises Tris:HCl, DMSO, DMEM, DPBS, or H2O.

86. The composition of claim 84, wherein the polymer is a sol-gel, gel, or solid.

87. The composition of claim 86, wherein the polymer is a hydrogel.

88. The composition of claim 87, wherein the hydrogel is a polysaccharide hydrogel, polyacrylamide hydrogel, polyacrylic acid hydrogel, polymethyl acrylate hydrogel, polyvinyl alcohol hydrogel, polyvinylpyrrolidone hydrogel, polyethylene glycol hydrogel, agarose hydrogel, gelatin hydrogel, collagen hydrogel, alginate hydrogel, or a combination thereof.

89. The composition according to claim 87, wherein the hydrogel is a polyacrylamide hydrogel or an agarose hydrogel.

90. The composition of claim 84, wherein the polymer is polystyrene, epoxy resin, polyvinyl acetate, ethylene-vinyl acetate, polyurethane, polytetrafluoroethylene (PTFE), polycarbonate, or polyvinyl butyral.

91. The composition of claim 83, wherein the uniform optical volume comprises fluorescent molecules.

92. The composition of claim 91, wherein the fluorescent molecule has an emission maximum of about 400 nm to about 720 nm.

93. The composition of claim 92, wherein the fluorescent molecule has an emission maximum of about 420 nm to about 480 nm.

94. The composition of claim 93, wherein the fluorescent molecule has an emission maximum of 450 nm.

95. The composition of claim 92, wherein the fluorescent molecule has an emission maximum of about 500 nm to about 550 nm.

96. The composition of claim 95, wherein the fluorescent molecule has an emission maximum of 517 nm.

97. The composition according to claim 96, wherein the fluorescent molecule is fluorescein.

98. The composition of claim 95, wherein the fluorescent molecule has an emission maximum of 525 nm.

99. The composition of claim 95, wherein the fluorescent molecule has an emission maximum of 545 nm.

100. The composition of claim 92, wherein the fluorescent molecule has an emission maximum of about 525 nm to about 575 nm.

101. The composition of claim 95, wherein the fluorescent molecule has an emission maximum of 550 nm.

102. The composition according to claim 101, wherein the fluorescent molecule is Rhodamine B.

103. The composition of claim 100, wherein the fluorescent molecule has an emission maximum of 565 nm.

104. The composition of claim 92, wherein the fluorescent molecule has an emission maximum of about 575 nm to about 650 nm.

105. The composition of claim 104, wherein the fluorescent molecule has an emission maximum of 585 nm.

106. The composition of claim 104, wherein the fluorescent molecule has an emission maximum of 605 nm.

107. The composition of claim 104, wherein the fluorescent molecule has an emission maximum of 625 nm.

108. The composition of claim 92, wherein the fluorescent molecule has an emission maximum of about 650 nm to about 720 nm.

109. The composition of claim 108, wherein the fluorescent molecule has an emission maximum of 655 nm.

110. The composition of claim 108, wherein the fluorescent molecule has an emission maximum of 705 nm.

111. The composition of claim 91, wherein the fluorescent molecule is a quenched fluorescent molecule.

112. The composition of claim 1, wherein the one or more resolution test patterns are suitable for evaluating one or more of a resolution test pattern, a field distortion test pattern, or a parfocal stability test pattern.

113. The composition of claim 1, wherein the one or more resolution test patterns are selected from absorptive patterns, reflective patterns, and birefringent patterns.

114. A method for evaluating multiple performance characteristics of a microscope, the method comprising: A. Align the objective lens of the microscope with one of a plurality of individually addressable reference samples of the composition according to any one of claims 1 to 113, wherein the vertical axis of the objective lens is perpendicular to the horizontal axis of the sample; as well as (i) Analyze one or more imaging parameters based on the images captured by the microscope; (ii) Analyze one or more acquisition parameters based on the images captured by the microscope; (iii) Analyze one or more single-molecule tracking (SMT) analysis parameters based on the images captured by the microscope; or (iv). Combinations of (i) to (iii); B. Repeat the alignment and analysis steps for multiple individually addressable reference samples; as well as C. Integrate the analyses performed on the plurality of individually addressable reference samples, thereby assessing the plurality of microscope performance characteristics.

115. The method of claim 114, wherein the imaging parameters include: Laser parameters, alignment parameters, camera parameters, or detection parameters, or a combination thereof.

116. The method of claim 115, wherein analyzing the laser parameters includes analyzing one or more of the laser wavelength, laser pulse, and laser pulse duration.

117. The method of claim 115, wherein analyzing the alignment parameters comprises analyzing one or both of the light sheet characteristics and the focal plane.

118. The method of claim 117, wherein analyzing the characteristics of the light sheet includes analyzing one or more of the following: light sheet thickness; light sheet uniformity; light sheet tilt angle; and intensity density of the light sheet.

119. The method of claim 116, wherein analyzing the focal plane includes analyzing the position, flatness, orientation, and / or thickness of the optical focal plane relative to the sample and detector.

120. The method of claim 115, wherein analyzing the camera parameters includes analyzing one or more of the camera's noise mode, relative alignment, orientation, or magnification.

121. The method of claim 115, wherein analyzing the detection parameters includes analyzing one or more of aberrations, sensitivity, and resolution.

122. The method of claim 121, wherein analyzing aberrations includes analyzing one or more of spherical aberration, chromatic aberration, coma aberration, and cloverleaf aberration.

123. The method according to claim 114, wherein analyzing the acquisition parameters includes analyzing time parameters and / or spatial parameters.

124. The method of claim 123, wherein analyzing the time parameters includes analyzing one or more of frame rate, exposure time, number of frames, or channels.

125. The method of claim 123, wherein analyzing the spatial parameters includes analyzing one or more of the following: the field of view (FOV) size on the detector chip, the FOV position on the detector chip, the FOV position in the sample, or the FOV orientation.

126. The method of claim 114, wherein analyzing the SMT parameters includes analyzing single-molecule localization and / or single-molecule tracking.

127. The method of claim 126, wherein analyzing the single-molecule localization includes analyzing one or more of localization error, spot count, or signal-to-noise ratio (SNR).

128. The method of claim 123, wherein analyzing the single-molecule tracking includes analyzing one or more of the following: the number of trajectories, the trajectory length, the jump length, or the average posterior diffusion coefficient.

129. The method of claim 114, wherein the microscope performance characteristics are selected from: oblique line scanning (OLS) alignment characteristics: rotation relative to the camera, position in the FOV, tilt angle, excitation flux; oblique line scanning (OLS) uniformity characteristics: Coefficient of variation (CV), sagitta, stripes, thickness, mechanical and temperature effects; Detection features: background and camera noise, point spread function (PSF), signal-to-noise ratio (SNR), diffusion characteristics, optical aberrations, correction ring settings, spatial resolution, Strell ratio, FOV uniformity, and camera rotation relative to the sample; Acquisition features: plate position and levelness, scan amplitude, scan offset, galvo scanning, and camera synchronization.

130. The method of any one of claims 114, wherein the method further comprises comparing a result of each of the imaging parameters, the acquisition parameters, and the single-molecule tracking (SMT) parameters with a reference parameter value, wherein a deviation from the reference parameter value indicates that the microscope needs to be adjusted.

131. The method of claim 130, wherein the method further comprises automatically adjusting the microscope based on the integrated analysis.

Images