# US 12,180,531 B2 · Analysis and screening of cell secretion profiles

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- Number: US 12,180,531 B2
- Document ID: US12180531B2
- Status: Granted
- Kind: B2
- Application number: 18/088,110
- Filed: 2022-12-23
- Published: 2023-05-04
- Granted: 2024-12-31
- Earliest claimed: 2014-12-03
- Assignees: IsoPlexis Corporation
- Applicants: IsoPlexis Corporation
- Inventors: Patrick Paczkowski; Sean Mackay; Sean Mccusker
- Web page: https://patentgrep.com/patent/US12180531B2

## CPC

- C12Q 1/00: Measuring or testing processes involving enzymes, nucleic acids or microorganisms (measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34); Compositions therefor; Processes of preparing such compositions
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- B01L 3/5085: {for multiple samples, e.g. microtitration plates}
- C12M 41/46: {of cellular or enzymatic activity or functionality, e.g. cell viability}
- G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- G01N 33/543: with an insoluble carrier for immobilising immunochemicals
- G06T 7/13: Edge detection
- G06T 7/30: Determination of transform parameters for the alignment of images, i.e. image registration
- B01L 2200/0668: Trapping microscopic beads
- G06T 2207/10064: Fluorescence image
- G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro
- G06T 2207/30204: Marker
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- 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)
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL

## Abstract

Embodiments disclose apparatus, methods and software for performing biological screening and analysis implemented using an instrument platform capable of detecting a wide variety of cell-based secretions, expressed proteins, and other cellular components. The platform may be configured for simultaneous multiplexed detection of a plurality biological components such that a large number of discrete samples may be individually sequestered and evaluated to detect or identify constituents from the samples in a highly parallelized and scalable manner.

## Claims

### Claim 1

A method for spatial analysis of analytes, the method comprising:
(a) placing a sample comprising a plurality of cells on a first substrate;
(b) contacting the sample with a second substrate, the second substrate comprising (i) a plurality of analyte detection regions patterned with a plurality of discretely positioned analyte detection moieties, and (ii) a plurality of alignment markers disposed about the plurality of analyte detection regions, wherein the analyte detection moieties selectively associate with analytes released from the plurality of cells;
(c) obtaining one or more images, the one or more images comprising at least one image of the plurality of cells and at least one image of the plurality of alignment markers;
(d) detecting the analytes that selectively associated with corresponding analyte detection moieties of the plurality of analyte detection regions;
(e) using the location of at least one of the plurality of alignment markers in the one or more images to interpolate positioning of the plurality of analyte detection regions in the one or more images; and
(f) overlaying signals associated with the detected analytes with the interpolated positions of the plurality of analyte detection regions and the plurality of cells in the one or more images to produce a composite representation of regions of overlap between the plurality of cells and the detected analytes.

### Claim 2 (depends on 1)

The method of claim 1, wherein the analytes comprise nucleic acids and the analyte detection moieties comprise nucleic acids.

### Claim 3 (depends on 1)

The method of claim 1, wherein the analyte detection moieties are arranged as a plurality of spots.

### Claim 4 (depends on 1)

The method of claim 1, wherein the analyte detection moieties are arranged as a plurality of lines.

### Claim 5 (depends on 1)

The method of claim 1, wherein the one or more images comprises a bright field image.

### Claim 6 (depends on 1)

The method of claim 1, wherein the number of alignment markers on the second substrate is redundant such that poorly resolved alignment markers in the image of the plurality of alignment markers can be excluded from further analysis.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first substrate comprises a plurality of microchambers.

### Claim 8

A system for spatial analysis of analytes associated with a cellular population comprising:
a plurality of sample retention regions that receive at least one cell from a population of cells and retain an associated plurality of analytes released by the at least one cell;
a boundary mask delineating the plurality of sample retention regions;
a plurality of analyte detection regions patterned with a plurality of discretely positioned analyte detection regions and a plurality of alignment markers disposed about the plurality of analyte detection regions, the analyte detection regions disposed in an ordered pattern alignable with the sample retention regions whereby, upon coupling, released analytes selectively associate with analyte detection moieties in the analyte detection regions;
an imaging apparatus that generates images of the sample retention regions delineated by the boundary mask and the analyte detection regions; and
an image processor that aligns images for selected sample retention regions with corresponding analyte detection regions using the boundary mask and the plurality of alignment markers, the image processor further identifying the at least one cell in respective sample retention regions and corresponding analyte patterns to discretely resolve released analytes associated with the at least one cell based on the analyte detection moieties in the analyte detection regions.

### Claim 9 (depends on 8)

The system of claim 8, wherein the image processor is configured to use the location of the plurality of alignment markers in the image to interpolate positioning of the plurality of analyte detection regions in the image.

### Claim 10 (depends on 9)

The system of claim 9, wherein the image processor is configured to overlay signals associated the analytes with the interpolated positions of the plurality of analyte detection regions and the at least one cell to produce a composite representation of regions of overlap between the at least one cell and the analytes.