Methods of chromogen separation-based image analysis

• US 8,492,156 B2
• Filed: 05/12/2006
• Issued: 07/23/2013
• Est. Priority Date: 05/13/2005
• Status: Active Grant

• Current Assignee: Tripath Imaging Incorporated (Becton, Dickinson & Co)
• Original Assignee: Tripath Imaging Incorporated (Becton, Dickinson & Co)
• Inventors: Marcelpoil, Raphal, Williams, Ryan, Orny, Cdrick
• Primary Examiner(s): Menon, Krishnan S
• Assistant Examiner(s): Fritchman, Rebecca M
• Application Number:
  US11/433,114
• Publication Number:
  US 20070026525A1
• Time in Patent Office: 2,629 Days
• Field of Search: 436/63, 382/128, 382/133
• US Class Current: 436/63
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 1/31 Apparatus therefor
• CPC Class Codes: G06T 2207/10016 Video; Image sequence
• CPC Class Codes: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/20152 Watershed segmentation
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/11 Region-based segmentation
• CPC Class Codes: G06T 7/136 involving thresholding
• CPC Class Codes: G06T 7/155 involving morphological operators
• CPC Class Codes: G06V 20/695 Preprocessing, e.g. image segmentation

Abstract

Methods for chromogen separation-based image analysis are provided, with such methods being directed to quantitative video-microscopy techniques in cellular biology and pathology applications.

Claims

1. A method of staining a sample for microscopy imaging whereby the image of the stained sample is configured to exhibit an optimum contrast between sub-cellular components for diagnosis by a pathologist, said method comprising:

staining a sample with a dye;

determining a transmittance value of the dye from a microscopy image of the sample;

forming an artificial image of the sample from the determined transmittance value of the dye;

varying the transmittance value of the dye, by applying one of an absolute weighting coefficient and a relative weighting coefficient to an optical density corresponding thereto, so as to form a series of artificial images each demonstrating a different transmittance value of the dye;

selecting one image, from the series of images, exhibiting the optimum contrast between sub-cellular components for the dye and determining the corresponding transmittance value of the dye in the one image; and

varying staining of the sample with the dye so as to provide a stained sample having the transmittance value of the dye corresponding to the optimum contrast between sub-cellular components.

2. A method according to claim 1 further comprising determining an amount of the dye required to stain the sample so as to provide a stained sample having the transmittance value of the dye corresponding to the optimum contrast between sub-cellular components.

3. A method according to claim 1 wherein determining a transmittance value of the dye further comprises determining a transmittance value of the dye in each of a red channel, a green channel, and a blue channel from the microscopy image of the sample.

7. A method according to claim 1, wherein varying staining comprises electronically staining the sample with the dye.

8. A method according to claim 1, wherein determining the transmittance value comprises determining a concentration of the dye, and wherein varying the transmittance value comprises varying the transmittance value of the dye after the concentration of the dye has been determined.

4. A method of artificially staining a sample, said method comprising:

staining a sample with a first dye and a second dye;

determining a transmittance value and an extinction coefficient of each of the first dye and the second dye, in each of a red channel, a green channel, and a blue channel of an RGB color space, from a microscopy image of the sample;

applying a weighting coefficient to the extinction coefficient of at least one of the first dye and the second dye, each weighting coefficient being selected to change a proportion of the at least one of the first dye and the second dye staining the sample by changing the corresponding transmittance value of the at least one of the first dye and the second dye; and

forming an artificial image of the sample from the changed transmittance value of the at least one of the first dye and the second dye having the weighting coefficient applied thereto, and any unchanged transmittance value of the first dye and the second dye, in each of the red, green, and blue channels of the RGB color space.

5. A method according to claim 4, further comprising substituting an extinction coefficient of another for the extinction coefficient of one of the first dye and the second dye, in each of the red, green, and blue channels of the RGB color space.

6. A method according to claim 4, further comprising:

providing a series of artificial images varying by the proportion of one of the first dye and the second dye staining the sample; and

evaluating the series of artificial images to determine the artificial image having the optimum contrast between the components of the sample.

9. A method according to claim 4, wherein determining the transmittance value comprises determining a concentration of the first and second dyes, and wherein applying the weighting coefficient comprises applying the weighting coefficient after the concentrations of the first and second dyes have been determined.