Methods of chromogen separation-based image analysis
First Claim
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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 so as to form a series of artificial images;
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.
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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.
38 Citations
38 Claims
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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:
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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 so as to form a series of artificial images;
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. - View Dependent Claims (2, 3, 4)
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5. A method of artificially staining a sample, said method comprising:
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staining a sample with a first dye;
determining a transmittance value and an extinction coefficient of the first dye from a microscopy image of the sample;
forming an artificial image of the sample from the determined transmittance value of the first dye; and
substituting an extinction coefficient of a second dye for the extinction coefficient of the first dye so as to artificially stain the sample with the second dye. - View Dependent Claims (6, 7)
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8. A method of obtaining measurements of a sample from an image thereof, comprising:
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selecting a region of interest in the sample from an RGB image thereof;
segmenting the region of interest in the RGB image to identify any objects of interest therein;
implementing feature extraction to determine measurements for the identified objects of interest; and
determining cell scores with respect to at least one of marker localization and signal to noise ratio. - View Dependent Claims (9, 10, 11, 12, 13)
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14. A method of selecting a region of interest on a slide, the region being positively contrasted from a surrounding thereof in a marker-only image corresponding to an RGB image of the sample, the positively contrasted region including at least one of a relatively larger nuclei and a relatively higher cell density than the surrounding, comprising:
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applying a low pass filter to a marker-only image of a sample, the marker-only image being obtained through chromogen separation of the RGB image of the sample;
determining a marker-only histogram of pixels in the marker-only image; and
binarizing the marker-only image according to a threshold in the marker-only histogram so as to form a mask for discriminating between negative and positive regions of the sample. - View Dependent Claims (15, 16, 17)
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18. A method of segmenting a sample from an image thereof, comprising:
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determining a background component of an RGB image of the sample via a thresholding process;
segmenting the image by creating a component image of at least one of a membrane, a cytoplasm, and a nucleus;
refining the segmented image; and
filtering any unwanted objects from the image. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A method of determining optical density data for at least one dye staining a sample, for a high dye concentration, from an image obtained with a low bit resolution imaging device, comprising:
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capturing a series of images of the sample at different integration times;
selecting a highest non-saturated intensity in each of a red, green, and blue channel of the imaging device; and
reconstructing an optimized image of the sample using the highest non-saturated intensity levels in the red, green, and blue channels such that the optimized image is suitable for chromogen separation. - View Dependent Claims (37)
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38. A chromogen separation method for an image of a biological sample stained with four dyes obtained with a three channel imaging device, comprising:
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defining a priori known significant three dye combinations of the four dyes spatially collocated in the biological sample;
obtaining an image of a sample stained with four dyes with an imaging device having a red, green, and blue channel, the image thereby including a plurality of pixels each having a corresponding RGB triplet;
projecting each RGB triplet onto an extinction coefficient plane where
Ecr+Ecg+Ecb=1;determining the three dye combination of the four dyes in the extinction coefficient plane corresponding to each RGB triplet; and
separating the image of the sample by tabulating an amount of pixels in the image corresponding to each three dye combination in the extinction coefficient plane.
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Specification