# US 9,435,738 B2 · System and device for characterizing cells

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- Number: US 9,435,738 B2
- Document ID: US9435738B2
- Status: Granted
- Kind: B2
- Application number: 14/560,759
- Filed: 2014-12-04
- Published: 2015-04-02
- Granted: 2016-09-06
- Earliest claimed: 2011-06-22
- Assignees: The Johns Hopkins University; University of Florida
- Applicants: Denis Wirtz; Pei-Hsun Wu; Shyam B. Khatau; Wei-Chang Chen; Jude M. Phillip, Jr.; Zev A. Binder; Yiider Tseng
- Inventors: Denis Wirtz; Pei-Hsun Wu; Shyam B. Khatau; Wei-Chang Chen; Jude M. Phillip, Jr.; Zev A. Binder; Yiider Tseng
- Web page: https://patentgrep.com/patent/US9435738B2

## CPC

- G06T 7/0012: {Biomedical image inspection}
- G01N 15/14: Optical investigation techniques, e.g. flow cytometry
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G01N 33/48728: {Investigating individual cells, e.g. by patch clamp, voltage clamp (investigating individual particles in general G01N15/10)}
- G01N 33/48792: {Data management, e.g. communication with processing unit (for in vivo diagnostics A61B5/0002; transmission systems for measured values G08C)}
- G06V 10/28: Quantising the image, e.g. histogram thresholding for discrimination between background and foreground patterns
- G06V 20/69: Microscopic objects, e.g. biological cells or cellular parts
- G06V 20/693: {Acquisition}
- G06V 20/698: {Matching; Classification}
- G01N 2201/06113: Coherent sources; lasers
- G01N 2201/10: Scanning
- G06T 2207/10056: Microscopic image
- G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- 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)
- G06V: IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING

## Abstract

A diagnostic device includes a microscope configured to obtain image data on a plurality of cells and a computing device. The computing device is configured to receive the image data, identify at least a portion of each of the plurality of cells based on the received image data, determine at least one of a value of a morphological parameter for each identified at least a portion of the plurality of cells or a relative organization among the identified at least a portion of the plurality of cells, and calculate statistics for the plurality of cells based on the at least one of the determined values of the morphological parameter or the determined relative organization, the statistics including information suitable for distinguishing metastatic cells from non-metastatic cells. The diagnostic device further includes an output device configured to output the statistics for diagnosis.

## Claims

### Claim 1

A microscopy device comprising:
a microscope configured to obtain image data on a plurality of cells in different cell cycle phases on a substrate;
a computing device configured to:
receive the image data;
normalize the received image data;
identify at least a portion of each of the plurality of cells based on the normalized image data;
determine a value of a morphological parameter for each identified at least a portion of each of the plurality of cells; and
determine values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases based on the determined value of the morphological parameter for each identified at least a portion of each of the plurality of cells; and
an output device configured to output the values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases,
wherein identifying at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting nuclei of the plurality of cells in the normalized image data,
wherein segmenting the at least a portion of each of the plurality of cells comprises:
obtaining Gaussian intensity image data of the normalized image data;
obtaining averaged intensity image data of the normalized image data;
obtaining nuclear intensity image data based on the difference between the Gaussian intensity image data and the averaged intensity image data; and
segmenting nuclei based on the nuclear intensity image data.

### Claim 2 (depends on 1)

The microscopy device of claim 1, wherein normalizing the received image data comprises:
obtaining dark image data from the microscope;
obtaining reference illuminated image data from the microscope;
calibrating the image data of the plurality of cells based on the difference between intensities in the image data of the plurality of cells and the dark image data and the difference between intensities in the dark image data and the reference illuminated image data.

### Claim 3

A microscopy device comprising:
a microscope configured to obtain image data on a plurality of cells in different cell cycle phases on a substrate;
a computing device configured to:
receive the image data;
normalize the received image data;
identify at least a portion of each of the plurality of cells based on the normalized image data;
determine a value of a morphological parameter for each identified at least a portion of each of the plurality of cells; and
determine values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases based on the determined value of the morphological parameter for each identified at least a portion of each of the plurality of cells; and
an output device configured to output the values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases,
wherein identifying at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting nuclei of the plurality of cells in the normalized image data,
wherein segmenting at least a portion of each of the plurality of cells based on the normalized image data further comprises segmenting cell boundaries of the plurality of cells in the normalized image data,
wherein said segmenting cell boundaries comprises:
obtaining averaged image data of the normalized image data;
obtaining an average background intensity of the averaged image data;
obtaining a noise in background intensity magnitude;
filtering the normalized image data based on the average background intensity and the noise in background intensity magnitude; and
segmenting cell boundaries based on the filtered normalized image data.

### Claim 4 (depends on 3)

The microscopy device of claim 3, wherein segmenting cell boundaries comprises:
segmenting cell boundaries based on watershed segmentation.

### Claim 5 (depends on 3)

The microscopy device of claim 3, wherein the image data of the plurality of cells comprises first fluorescence channel data and second fluorescence channel data,
wherein said segmenting nuclei of the cells in the normalized image data is based on the first fluorescence channel data,
wherein said segmenting cell boundaries in the normalized image data is based on the second fluorescence channel data.

### Claim 6

A method for determining morphological parameters of a plurality of cells:
receiving image data on the plurality of cells in different cell cycle phases on a substrate;
normalizing the received image data;
identifying at least a portion of each of the plurality of cells based on the normalized image data;
determining a value of a morphological parameter for each identified at least a portion of each of the plurality of cells;
determining values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases based on the determined value of the morphological parameter for each identified at least a portion of each of the plurality of cells; and
outputting the values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases,
wherein identifying at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting nuclei of the plurality of cells in the normalized image data and
wherein segmenting the at least a portion of each of the plurality of cells further comprises:
obtaining Gaussian intensity image data of the normalized image data;
obtaining averaged intensity image data of the normalized image data;
obtaining nuclear intensity image data based on the difference between the Gaussian intensity image data and the averaged intensity image data; and
segmenting nuclei based on the nuclear intensity image data.

### Claim 7

A microscopy device comprising:
a microscope configured to obtain image data on a plurality of cells in different cell cycle phases on a substrate;
a computing device configured to:
receive the image data;
normalize the received image data;
identify at least a portion of each of the plurality of cells based on the normalized image data;
determine a value of a morphological parameter for each identified at least a portion of each of the plurality of cells; and
determine values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases based on the determined value of the morphological parameter for each identified at least a portion of each of the plurality of cells; and
an output device configured to output the values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases,
wherein identifying at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting nuclei of the plurality of cells in the normalized image data,
wherein segmenting at least a portion of each of the plurality of cells based on the normalized image data further comprises segmenting cell boundaries of the plurality of cells in the normalized image data,
wherein the image data of the plurality of cells comprises first fluorescence channel data and second fluorescence channel data,
wherein said segmenting nuclei of the cells in the normalized image data is based on the first fluorescence channel data,
wherein said segmenting cell boundaries in the normalized image data is based on the second fluorescence channel data,
wherein segmenting at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting nuclei of the plurality of cells in the normalized image data,
wherein segmenting the at least a portion of each of the plurality of cells further comprises:
obtaining Gaussian intensity image data of the normalized image data;
obtaining averaged intensity image data of the normalized image data;
obtaining nuclear intensity image data based on the difference between the Gaussian intensity image data and the averaged intensity image data; and
segmenting nuclei based on the nuclear intensity image data.

### Claim 8

A microscopy device comprising:
a microscope configured to obtain image data on a plurality of cells in different cell cycle phases on a substrate;
a computing device configured to:
receive the image data;
normalize the received image data;
identify at least a portion of each of the plurality of cells based on the normalized image data;
determine a value of a morphological parameter for each identified at least a portion of each of the plurality of cells; and
determine values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases based on the determined value of the morphological parameter for each identified at least a portion of each of the plurality of cells; and
an output device configured to output the values of the morphological parameter for corresponding cell cycle phases and fractions of the plurality of cells in the corresponding cell cycle phases,
wherein identifying at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting nuclei of the plurality of cells in the normalized image data,
wherein segmenting at least a portion of each of the plurality of cells based on the normalized image data further comprises segmenting cell boundaries of the plurality of cells in the normalized image data,
wherein the image data of the plurality of cells comprises first fluorescence channel data and second fluorescence channel data,
wherein said segmenting nuclei of the cells in the normalized image data is based on the first fluorescence channel data,
wherein said segmenting cell boundaries in the normalized image data is based on the second fluorescence channel data,
wherein segmenting at least a portion of each of the plurality of cells based on the normalized image data comprises segmenting nuclei of the plurality of cells in the normalized image data
wherein segmenting at least a portion of each of the plurality of cells based on the normalized image data further comprises segmenting cell boundaries of the plurality of cells in the normalized image data,
wherein said segmenting cell boundaries comprises:
obtaining averaged image data of the normalized image data;
obtaining an average background intensity of the averaged image data;
obtaining a noise in background intensity magnitude;
filtering the normalized image data based on the average background intensity and the noise in background intensity magnitude; and
segmenting cell boundaries based on the filtered normalized image data.

### Claim 9 (depends on 8)

The microscopy device of claim 8, wherein segmenting cell boundaries comprises:
segmenting cell boundaries based on watershed segmentation.