# US 12,181,650 B2 · Methods and systems for three-dimensional lightsheet imaging > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,181,650 B2 - Document ID: US12181650B2 - Status: Granted - Kind: B2 - Application number: 17/836,737 - Filed: 2022-06-09 - Published: 2023-02-02 - Granted: 2024-12-31 - Earliest claimed: 2019-12-10 - Assignees: Enumerix, Inc. - Applicants: Enumerix, Inc. - Inventors: Janice Lai; David Stern - Web page: https://patentgrep.com/patent/US12181650B2 ## CPC - G02B 21/0032: {Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers (G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06)} - G01N 15/1429: Signal processing - G01N 15/1434: Optical arrangements - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G02B 21/0036: {Scanning details, e.g. scanning stages} - G02B 21/0076: {arrangements using fluorescence or luminescence} - G02B 21/008: {Details of detection or image processing, including general computer control} - G02B 21/26: Stages; Adjusting means therefor - G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates} - G01N 2015/1445: {Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography} - G01N 2015/1497: {Particle shape} - G01N 2021/6482: {Sample cells, cuvettes} - G01N 2201/129: Using chemometrical methods - G02B 3/06: with cylindrical or toric faces - G02B 3/08: with discontinuous faces, e.g. Fresnel lens {(diffractive Fresnel lenses G02B5/1876)} - G02B 27/30: Collimators - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Disclosed herein are light sheet imaging systems for imaging fluorescent samples. Also disclosed herein are sample holder systems for high throughput light sheet imaging of multiple three-dimensional samples without user intervention. Further disclosed herein are automated image processing methods to identify and quantify fluorescent particles within three-dimensional image sets without user intervention or user bias. ## Claims ### Claim 1 A light sheet imaging system comprising: a plurality of containers comprising a plurality of samples, wherein each sample of the plurality of samples comprises a distribution of fluorescent particles and is contained within a container of the plurality of containers; a collimated illumination source configured to emit a collimated beam along a beam path; a Powell lens positioned in the beam path after the collimated illumination source and configured to expand the collimated beam along a single axis; a first cylindrical lens positioned in the beam path after the Powell lens and configured to re-collimate the beam along a single axis, thereby producing a collimated beam having a short axis orthogonal to a direction of propagation and long axis orthogonal to the short axis and the direction of propagation; and a second cylindrical lens positioned in the beam path after the first cylindrical lens and configured to focus the beam along the short axis to a focal plane; wherein the light sheet imaging system is configured to scan a set of cross-sections of each sample of the plurality of samples within each container of the plurality of containers. ### Claim 2 (depends on 1) The light sheet imaging system of claim 1, wherein the plurality of samples comprises a three-dimensional sample comprising a distribution of fluorescently-labeled material positioned at the focal plane. ### Claim 3 (depends on 1) The light sheet imaging system of claim 1, further comprising a camera comprising an imaging path orthogonal to the beam path, wherein the camera is positioned such that the imaging path intersects the beam path at the focal plane. ### Claim 4 (depends on 1) The light sheet imaging system of claim 1, further comprising a sample holder, wherein the sample holder comprises: a fluid reservoir configured to contain a fluid, the fluid reservoir comprising a base, a first transparent side, a second transparent side connected to the first transparent side at a right angle, and a third transparent side opposite the first transparent side and connected to the first transparent side and the second transparent side; a tube holder configured to hold a row of a plurality of tubes partially submerged in the fluid; and a translating stage supporting the fluid reservoir and the tube holder and configured to translate the fluid reservoir and the tube holder along a first translation axis and a second translation axis, wherein the first translation axis is oriented parallel to the first transparent side and perpendicular to the second transparent side, and wherein the second translation axis is perpendicular to the first transparent side and parallel to the second transparent side, and wherein the first translation axis and the second translation axis are oriented at a 45° angle relative to the row of the plurality of tubes. ### Claim 5 (depends on 4) The sample holder of claim 4, further comprising a fourth transparent side connected to the second transparent side at a right angle and parallel to the first transparent side, and a fifth transparent side connected to the fourth transparent side at a right angle and parallel to the second transparent side. ### Claim 6 (depends on 4) The sample holder of claim 4, wherein the plurality of tubes comprises at least one of PCR tubes and microcentrifuge tubes. ### Claim 7 A method comprising: generating a count of signal positive particles of a distribution of fluorescent particles, upon: collecting a three-dimensional image data set comprising a plurality of cross-section images of a container containing the distribution of fluorescent particles, the plurality of cross-section images generated upon scanning the container with a light sheet at a frequency from 0.1 to 20 kilohertz; identifying a plurality of substantially spherical fluorescent particles in the three-dimensional image data set; dividing one or more cross-section images of the plurality of cross-section images into a plurality of image grids; applying an intensity threshold independently to one or more image grids of the plurality of image grids; and identifying one or more signal positive particles from the plurality of substantially spherical fluorescent particles, wherein the one or more signal positive particles have intensities greater than the intensity threshold. ### Claim 8 (depends on 7) The method of claim 7, wherein identifying the plurality of substantially spherical fluorescent particles comprises performing a convolution using a template. ### Claim 9 (depends on 7) The method of claim 7, further comprising determining an intensity of one or more substantially spherical fluorescent particles of the plurality of substantially spherical fluorescent particles by a three-dimensional local maxima analysis, and performing a second three-dimensional maxima analysis to remove one or more asymmetric particles from the plurality of substantially spherical fluorescent particles. ### Claim 10 (depends on 7) The method of claim 7, further comprising: determining a ratio of a number of signal positive particles to a number of the plurality of substantially spherical florescent particles for an image grid of the plurality of image grids, identifying the ratio as a ratio outlier if the ratio is outside of a threshold distance from a median of ratios determined from at least a subset of image grids of the plurality of image grids, and applying a second intensity threshold to the image grid of the plurality of image grids to determine a refined ratio for the image grid. ### Claim 11 (depends on 7) The method of claim 7, further comprising selecting a subset of the plurality of substantially spherical fluorescent particles, wherein the subset of the plurality of substantially spherical fluorescent particles comprise a size larger than a minimum size threshold and smaller than a maximum size threshold. ### Claim 12 (depends on 7) The method of claim 7, further comprising: determining a ratio of a number of signal positive particles to a number of the plurality of substantially spherical fluorescent particles for an image grid of the plurality of image grids, identifying the ratio as a ratio outlier if the ratio is outside of a threshold distance from a median of ratios determined from at least a subset of image grids of the plurality of image grids, and removing the ratio outlier. ### Claim 13 (depends on 7) The method of claim 7, further comprising performing cluster removal to eliminate overlapping fluorescent particles. ### Claim 14 (depends on 13) The method of claim 13 wherein the cluster removal comprises: determining a number of close droplet neighbors for a plurality of particles of the fluorescent particles; generating a histogram of close particle neighbors for the plurality of particles; and eliminating outlier particles having a number of close particle neighbors over a set threshold, wherein the set threshold is based at least in part upon a probability that a particle of the plurality of particles is a false signal positive particle merged with a signal negative particle. ### Claim 15 A method of analyzing a plurality of samples, the plurality of samples comprising at least 24 samples each comprising a distribution of fluorescent particles, the method comprising automatically scanning a set of cross-sections of each sample of the plurality of samples with a laser beam, thereby generating emission light in each sample of the plurality of samples, wherein the laser beam is a sheet of laser light. ### Claim 16 (depends on 15) The method of claim 15, wherein the plurality of samples is located in a sample holder, and wherein automatically scanning the set of cross-sections comprises moving the sample holder in a direction perpendicular to an illumination path of the laser beam to move a scanned first sample of the plurality of samples out of the illumination path of the laser beam and to move a second sample of the plurality of samples into the illumination path of the laser beam. ### Claim 17 (depends on 16) The method of claim 16, further comprising, while automatically scanning the set of cross-sections, reducing image artifacts in resulting image data generated from the plurality of samples, by moving the laser beam relative to the sample with at least one of pivoting movements and translating movements of the laser beam. ### Claim 18 (depends on 17) The method of claim 17, wherein the laser beam moves relative to the sample with a frequency of from about 0.1 kHz to about 20 kHz. ### Claim 19 (depends on 15) The method of claim 15, wherein the plurality of samples is subjected to a second laser beam opposing the laser beam.