Stain unmixing of multiplexed brightfield images

A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform actions including:

• determining, for each stain of at least three digital pathology stains, a color vector that represents the stain;
• accessing a real multiplex digital pathology image that depicts a biopsy section stained with at least one first stain of the at least three stains, wherein the depicted biopsy section is not stained with at least one second stain of the at least three stains;
• generating a filtered output by filtering the real multiplex digital pathology image using the color vector that represents a second stain of the at least one second stain;
• generating a metric that characterizes a signal characteristic in the filtered output;
• using the metric and a space-traversal technique to identify an adjustment of the color vector that represents the second stain;
• receiving a new multiplex image stained with at least one of the at least three digital pathology stains;
• generating a new synthetic singleplex image based on the new multiplex image and the adjusted color vector that represents the second stain;
• and
• outputting the new synthetic singleplex image.

A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform actions including:

• determining, for each stain of at least two digital pathology stains, a color vector that represents the stain;
• accessing a real multiplex digital pathology image that depicts a biopsy section stained with the at least two digital pathology stains;
• identifying a recommended color vector that represents a potential additional stain by:
• identifying an initial color vector;
• generating a filtered output by filtering the real multiplex digital pathology image using the initial color vector;
• generating a metric that characterizes a signal characteristic in the filtered output;
• and
• using the metric and a space-traversal technique to identify the recommended color vector;
• and
• outputting the recommended color vector.

A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform actions including:

• determining, for each stain of at least three digital pathology stains, a color vector that represents the stain;
• accessing a real multiplex digital pathology image that depicts a biopsy section stained with at least one first stain of the at least three digital pathology stains, wherein the depicted biopsy section is not stained with at least one second stain of the at least three stains;
• generating a filtered output by filtering the real multiplex digital pathology using the color vector that represents a second stain of the at least one second stain;
• generating a performance-prediction score that represented a predicted extent to which the at least three digital pathology stains are sufficiently separable in practice to reliably support generation of synthetic singleplex images;
• and
• outputting the performance-prediction score.

A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform actions including:

• determining, for each stain of at least four digital pathology stains, a color vector that represents the stain;
• wherein the determined color vectors are within a multi-dimensional color space,
• selecting a specific stain of the at least four digital pathology stains;
• determining a portion of the color space that is predicted to be attributable to prominent signals that correspond to a the specific stain;
• accessing a real multiplex digital pathology image that depicts a biopsy section stained with at least three digital pathology stains of the at least four digital pathology stains, wherein the real multiplex digital pathology image includes a set of pixels;
• mapping each pixel of the set of pixels in the real multiplex digital pathology image to a point within the multi-dimensional color space;
• generating, for each pixel of the set of pixels, a pixel-specific color vector that predicts, for each of the at least four digital pathology stains, a degree of expression of the stain in a part of the biopsy section that is depicted at the pixel, wherein generating the pixel-specific color vectors includes:
• determining that each of a first subset of the set of pixels is mapped to a point that is within the portion of the color space;
• determining, for each pixel of the first subset of pixels, an optical density, wherein the pixel-specific color vector for the pixel identifies a degree of expression for the specific stain that corresponds to the optical density;
• determining that each of a second subset of the set of pixels is mapped to a point that is outside of the portion of the color space;
• and
• performing an unmixing technique to predict, for each pixel in the second subset and for each of some of the at least four digital pathology stains, a degree of expression of the stain in the part of the biopsy section that is depicted at the pixel, wherein the some of the at least four digital pathology stains does not include the specific stain, and wherein the unmixing technique uses the color vector determined to represent each of the some of the at least four digital pathology stains;
• and
• generating one or more synthetic singleplex images using the pixel-specific color vectors.