Spatial multiplexing of histological stains

Abstract

The following concerns a method for co-localization of microscopy or histology stains by the assembly of a virtual image from one or more imaging operations. In particular, the method decreases the time required to obtain multiple labeled antigen or protein histology images of a biological sample. The method includes imaging the tissue as it is sliced by a microtome with a knife edge scanning microscope and spatially aligning the samples by the generated images. The spatial alignment of samples enabled by the method allows a panel of different antigen or protein secondary or functional stains to be compared across different sample slices, thereby allowing concurrent secondary stains of tissues and cells.

What is claimed is: 
     
         1 . A method of analyzing a sample, the method comprising:
 slicing the sample into a plurality of sections;   for each section in the plurality of sections:
 generating a primary image of the section; 
 associating the primary image with the section; 
 based on information from the primary image of the section, determining whether to stain the section; 
 based on a determination that the section is to be stained:
 determining a staining agent to be used to stain the section using the information from the primary image of the section; 
 staining the section using the staining agent; 
 generating a secondary image of the section by imaging the stained section; and 
 associating the secondary image with the section. 
 
   
     
     
         2 . The method of  claim 1 , wherein the sample comprises a tissue sample. 
     
     
         3 . The method of  claim 1 , further comprising:
 for each section in the plurality of sections having an associated primary image and an associated secondary image:
 co-registering the associated primary image with the associated secondary image by post-processing the associated primary image having no distortions with the associated secondary image having distortions that occurred during the staining, thereby improving post-processing of images; and 
 wherein the post-processing includes processing distortions in the associated secondary image based on the associated primary image; and 
   co-registering the secondary images associated with the plurality of sections with one another.   
     
     
         4 . The method of  claim 1 , further comprising:
 generating a virtual model of the sample based on primary images associated with each section of the plurality of sections.   
     
     
         5 . The method of  claim 1 , further comprising:
 for each section in the plurality of sections having an associated primary image and an associated secondary image:
 co-registering the associated primary image with the associated secondary image by post-processing the associated primary image having no distortions with the associated secondary image having distortions that occurred during the staining, thereby improving post-processing of images; and 
 wherein the post-processing includes processing distortions in the associated secondary image based on the associated primary image; 
   co-registering the secondary images associated with the plurality of sections with one another; and   co-registering the co-registered plurality of secondary images of the sample to the virtual model.   
     
     
         6 . The method of  claim 5 , further comprising:
 generating a diagnosis in response to the co-registration of the co-registered plurality of secondary images of the sample and the virtual model.   
     
     
         7 . The method of  claim 1 , wherein slicing the sample into a plurality of sections comprises slicing a block face of the sample. 
     
     
         8 . The method of  claim 1 , further comprising:
 mounting the plurality of sections onto a plurality of slides.   
     
     
         9 . The method of  claim 1 , wherein the staining agent is one of: an antigen or protein stain. 
     
     
         10 . One or more non-transitory computer-readable storage media, storing one or more sequences of instructions, which when executed by one or more processors cause performance of:
 slicing the sample into a plurality of sections;   for each section in the plurality of sections:
 generating a primary image of the section; 
 associating the primary image with the section; 
 based on information from the primary image of the section, determining whether to stain the section; 
 based on a determination that the section is to be stained:
 determining a staining agent to be used to stain the section using the information from the primary image of the section; 
 staining the section using the staining agent; 
 generating a secondary image of the section by imaging the stained section; and 
 associating the secondary image with the section. 
 
   
     
     
         11 . The one or more non-transitory computer-readable storage media of  claim 10 , wherein the sample comprises a tissue sample. 
     
     
         12 . The one or more non-transitory computer-readable storage media of  claim 10 , further comprising:
 for each section in the plurality of sections having an associated primary image and an associated secondary image:
 co-registering the associated primary image with the associated secondary image by post-processing the associated primary image having no distortions with the associated secondary image having distortions that occurred during the staining, thereby improving post-processing of images; and 
 wherein the post-processing includes processing distortions in the associated secondary image based on the associated primary image; and 
   co-registering the secondary images associated with the plurality of sections with one another.   
     
     
         13 . The one or more non-transitory computer-readable storage media of  claim 10 , further comprising:
 generating a virtual model of the sample based on primary images associated with each section of the plurality of sections.   
     
     
         14 . The one or more non-transitory computer-readable storage media of  claim 10 , further comprising:
 for each section in the plurality of sections having an associated primary image and an associated secondary image:
 co-registering the associated primary image with the associated secondary image by post-processing the associated primary image having no distortions with the associated secondary image having distortions that occurred during the staining, thereby improving post-processing of images; and 
 wherein the post-processing includes processing distortions in the associated secondary image based on the associated primary image; 
   co-registering the secondary images associated with the plurality of sections with one another; and   co-registering the co-registered plurality of secondary images of the sample to the virtual model.   
     
     
         15 . The one or more non-transitory computer-readable storage media of  claim 14 , further comprising generating a diagnosis in response to the co-registration of the co-registered plurality of secondary images of the sample and the virtual model. 
     
     
         16 . The one or more non-transitory computer-readable storage media of  claim 10 , wherein slicing the sample into a plurality of sections comprises slicing a block face of the sample. 
     
     
         17 . The one or more non-transitory computer-readable storage media of  claim 10 , further comprising:
 mounting the plurality of sections onto a plurality of slides.   
     
     
         18 . The one or more non-transitory computer-readable storage media of  claim 10 , wherein the staining agent is one of: an antigen or protein stain. 
     
     
         19 . A method of analyzing a sample, the method comprising:
 slicing the sample into a plurality of sections;   for each section in the plurality of sections:
 generating a primary image of the section; 
 associating the primary image with the section; and 
 based on information from the primary image of the section, diverting the section to a molecular diagnostic.