Compartment segregation by pixel characterization using image data clustering
First Claim
1. A method for defining a first marker-defined biological compartment relative to a second marker-defined biological compartment present in a biological sample of interest, comprisingcomparing the intensity in each of the pixel locations in a first image of the first marker defined biological compartment with the intensity in each of the corresponding pixel locations of a second image of the second marker-defined biological compartment, wherein the first image was prepared using a first imaging agent that is specific for the first marker-defined biological compartment, and wherein the second image was prepared using a second imaging agent that is specific for the second marker-defined biological compartment, wherein differences in pixel intensity define the first marker-defined biological compartment relative to the second marker-defined biological compartment,assigning pixels to a cluster characterized by high first imaging agent intensity and low second imaging agent intensity to the first compartment;
- assigning pixels to a cluster characterized by high second imaging agent intensity and low first imaging agent intensity to the second compartment; and
assigning pixels to a cluster characterized by low first imaging agent intensity and low second imaging agent intensity to background and removing such pixels from further analysis,wherein any of the assigning steps are performed using a k means clustering method to determine a cluster membership for each pixel, andwherein the method is automated.
2 Assignments
0 Petitions
Accused Products
Abstract
The present invention relates generally to improved methods of defining areas or compartments within which biomarker expression is detected and quantified. In particular, the present invention relates to automated methods for delineating marker-defined compartments objectively with minimal operator intervention or decision making. The method provides for precise definition of tissue, cellular or subcellular compartments particularly in histological tissue sections in which to quantitatively analyzing protein expression.
-
Citations
14 Claims
-
1. A method for defining a first marker-defined biological compartment relative to a second marker-defined biological compartment present in a biological sample of interest, comprising
comparing the intensity in each of the pixel locations in a first image of the first marker defined biological compartment with the intensity in each of the corresponding pixel locations of a second image of the second marker-defined biological compartment, wherein the first image was prepared using a first imaging agent that is specific for the first marker-defined biological compartment, and wherein the second image was prepared using a second imaging agent that is specific for the second marker-defined biological compartment, wherein differences in pixel intensity define the first marker-defined biological compartment relative to the second marker-defined biological compartment, assigning pixels to a cluster characterized by high first imaging agent intensity and low second imaging agent intensity to the first compartment; -
assigning pixels to a cluster characterized by high second imaging agent intensity and low first imaging agent intensity to the second compartment; and assigning pixels to a cluster characterized by low first imaging agent intensity and low second imaging agent intensity to background and removing such pixels from further analysis, wherein any of the assigning steps are performed using a k means clustering method to determine a cluster membership for each pixel, and wherein the method is automated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
-
-
12. A computer implemented method for defining a first marker-defined biological compartment relative to a second marker-defined biological compartment present in a biological sample comprising:
-
a) incubating the sample with a first imaging agent that specifically labels the first marker-defined compartment, and a second imaging agent that specifically labels the second marker defined compartment; b) obtaining a first image of the first imaging agent labeled sample, and a second image of the second imaging agent labeled sample; c) determining a first and a second imaging agent intensity in each corresponding pixel location in the first and the second image; d) performing a clustering analysis on each pixel based on the first and the second imaging agent intensity of each pixel in each of the pixel to calculate clusters; e) assigning those pixels in the cluster characterized by high first imaging agent pixel intensity and low second imaging agent pixel intensity to the first compartment; f) assigning those pixels in the cluster characterized by high second imaging agent pixel intensity and low first imaging agent pixel intensity to the second compartment; and g) assigning those pixels in the cluster characterized by low first imaging agent pixel intensity and low second imaging agent pixel intensity to background and removing such pixels from further analysis, thereby defining a first marker-defined sub-cellular compartment relative to a second marker defined-sub-cellular compartment, wherein any of the assigning steps are performed using a k means clustering method to determine a cluster membership for each pixel, and wherein the method is automated.
-
-
13. A computer implemented method for defining a first marker defined sub-cellular compartment relative to a second marker defined sub-cellular compartment present in individual cells of interest contained in a tissue sample comprising:
-
a) incubating the tissue sample with a first stain that specifically labels the first marker defined sub-cellular compartment, a second stain that specifically labels a second marker defined sub-cellular compartment, b) obtaining an image of each of the first and the second stain in the tissue sample using a microscope so as to obtain; i) a first image of the first marker defined sub-cellular compartment; ii) a second image of the second marker defined sub-cellular compartment; and c) determining the first and second stain intensity in each of the pixel locations in the first and the second image; d) plotting the pixels, where the axes of the plot comprise the intensity of the first stain and the intensity of the second stain; e) performing reiterative k-means clustering on the first and the second stain intensity in each of the pixel locations to calculate three clusters; f) assigning those pixels in the cluster characterized by high first stain intensity and low second stain intensity to the first compartment; g) assigning those pixels in the cluster characterized by high second stain intensity and low first stain intensity to the second compartment; h) assigning those pixels in the cluster characterized by low first stain intensity and low second stain intensity to background and removing such pixels from further analysis; i) assigning those pixels with first stain intensity and second stain intensity to either the first compartment or the second compartment based upon based on probability thereby defining a first marker defined sub-cellular compartment relative to a second marker defined sub-cellular compartment, wherein any of the assigning steps are performed using a k means clustering method to determine a cluster membership for each pixel, and wherein the method is automated.
-
-
14. A computer implemented method for defining a first marker defined sub-cellular compartment relative to a second marker defined sub-cellular compartment present in individual cells of interest contained in a tissue sample comprising:
-
a) incubating the tissue sample with a first stain that specifically labels the first marker defined sub-cellular compartment, a second stain that specifically labels a second marker defined sub-cellular compartment, b) obtaining an image of each of the first and the second stain in the tissue sample using a microscope so as to obtain; i) a first image of the first marker defined sub-cellular compartment; ii) a second image of the second marker defined sub-cellular compartment; and c) determining the first and second stain intensity in each of the pixel locations in the first and the second image; d) plotting the pixels, where the axes of the plot comprise the intensity of the first stain and the intensity of the second stain; e) performing reiterative k-means clustering on the first and the second stain intensity in each of the pixel locations to calculate three clusters; f) assigning those pixels in the cluster characterized by high first stain intensity and low second stain intensity to the first compartment; g) assigning those pixels in the cluster characterized by high second stain intensity and low first stain intensity to the second compartment; h) assigning those pixels in the cluster characterized by low first stain intensity and low second stain intensity to background and removing such pixels from further analysis; i) assigning those pixels with first stain intensity and second stain intensity to neither the first compartment or the second compartment; thereby defining a first marker defined sub-cellular compartment relative to a second marker defined sub-cellular compartment, wherein any of the assigning steps are performed using a k means clustering method to determine a cluster membership for each pixel, and wherein the method is automated.
-
Specification