# US 8,571,294 B2 · Method for imaging and differential analysis of cells

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- Number: US 8,571,294 B2
- Document ID: US8571294B2
- Status: Granted
- Kind: B2
- Application number: 13/302,941
- Filed: 2011-11-22
- Published: 2012-04-26
- Granted: 2013-10-29
- Earliest claimed: 2004-03-16
- Assignees: Amnis Corporation
- Applicants: Thaddeus C. George; David A. Basiji; Brian E. Hall; William E. Ortyn; Michael E. Seo; Phillip J. Morrissey; Cathleen A. Zimmerman
- Inventors: Thaddeus C. George; David A. Basiji; Brian E. Hall; William E. Ortyn; Michael E. Seo; Phillip J. Morrissey; Cathleen A. Zimmerman
- Web page: https://patentgrep.com/patent/US8571294B2

## CPC

- G06V 20/69: Microscopic objects, e.g. biological cells or cellular parts
- G01N 21/47: Scattering, i.e. diffuse reflection (G01N21/25, G01N21/41 take precedence {G01N21/55 takes precedence})
- G01N 21/645: {Specially adapted constructive features of fluorimeters}
- G06V 20/698: {Matching; Classification}
- G01N 15/1433: using image recognition
- G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging}
- G01N 2021/6423: {Spectral mapping, video display}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- G01N 2021/6482: {Sample cells, cuvettes}
- G06V: IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
- 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

Provided are methods for determining and analyzing photometric and morphometric features of small objects, such as cells to, for example, identify different cell states. In particularly, methods are provided for identifying apoptotic cells, and for distinguishing between cells undergoing apoptosis versus necrosis.

## Claims

### Claim 1

A method for identifying a specific cell as being of a known cell type, wherein the known cell type relates to the viability of the cell, comprising:
staining a nucleus of the specific cell with a fluorescence stain;
directing incident light at the specific cell to obtain a nuclear image, a side scatter image and a brightfield image of the specific cell with a light detector; and
determining the known cell type to which the specific cell corresponds, based on morphologic features of the specific cell that are derived from the nuclear image, the brightfield image and the side scatter image, wherein the morphologic features include at least one of the cellular size, the nuclear size, the cytoplasmic size, and internal cell granularity of the specific cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cytoplasmic size is determined by subtracting an area of the nucleus of the specific cell that was stained from a total area of the brightfield image of the specific cell.

### Claim 3 (depends on 1)

The method of claim 1, wherein the internal cell granularity of the specific cell is determined based upon a spatial scatter frequency of data derived from the side scatter image of the specific cell.

### Claim 4 (depends on 1)

The method of claim 1, wherein the specific cell is stained with a nucleic acid-binding dye consisting of one of 7-aminoactinomycin D (7-AAD), and propidium iodide.

### Claim 5 (depends on 1)

The method of claim 1, wherein there is relative motion between the specific cell and the detector.

### Claim 6 (depends on 1)

The method of claim 1, wherein the specific cell identified is contained within a heterogeneous cell population, and the nuclear image, the brightfield image, and the side scatter image are collected for the specific cell from nuclear images, brightfield images, and side scatter images of the heterogeneous cell population.

### Claim 7 (depends on 1)

The method of claim 1, wherein the specific cell identified is an apoptotic cell.

### Claim 8 (depends on 7)

The method of claim 7, wherein the apoptotic cell is an early stage apoptotic cell or a late stage apoptotic cell.

### Claim 9 (depends on 1)

The method of claim 1, wherein the specific cell identified is a necrotic cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein the specific cell identified is at least one of an apoptotic cell and a necrotic cell.