Abstract: Automated methods and devices that facilitate iterative staining of biological samples from imaging applications are provided. The methods include the steps of providing a small volume flow cell containing a biological sample, applying a stain to the biological sample, combining at least two precursor reagents to form an activated destaining agent and wherein the activated destaining agent decomposition rate is greater than or similar to the destaining reaction rate, flowing the destaining agent over the biological sample at a flow rate that is greater than the decomposition rate of the activated destaining agent, and releasing the sample from the flow cell wherein the integrity of the sample is intact. The process of staining, combining and flowing may be iteratively repeated.

Abstract: The present techniques provide fully automated methods for quantifying the location, strength and percent of expressed target molecules or other biological markers in immunohistochemically stained biological samples. The samples may be automatically segmented, for example into subcellular compartments, from images of compartmental markers. Then, the distribution of a target molecule on each of these compartments is calculated that includes the percentage and strength of expression. This is different than existing intensity or ratio based methods where abundant low expression levels are indistinguishable from scarce high expression levels.

Abstract: Techniques for removing image autoflourescence from fluorescently stained biological images are provided herein. The techniques utilize non-negative matrix factorization that may constrain mixing coefficients to be non-negative. The probability of convergence to local minima is reduced by using smoothness constraints. The non-negative matrix factorization algorithm provides the advantage of removing both dark current and autofluorescence.

Abstract: The invention provides a method for antigen retrieval of a formaldehyde-fixed tissue sample comprising incubating a formaldehyde-fixed tissue sample in a first antigen retrieval solution at a temperature of greater than 90° C., transferring the tissue sample to a second antigen retrieval solution, and incubating the tissue sample in the second antigen retrieval solution at a temperature of greater than 90° C. The invention also provides a kit and sample delivery device for carrying out the method.

Abstract: The invention provides a method for antigen retrieval of a formaldehyde-fixed tissue sample comprising incubating a formaldehyde-fixed tissue sample in a first antigen retrieval solution at a temperature of greater than 90° C., transferring the tissue sample to a second antigen retrieval solution, and incubating the tissue sample in the second antigen retrieval solution at a temperature of greater than 90° C. The invention also provides a kit and sample delivery device for carrying out the method.

Abstract: Method and systems for removing any inherent autofluorescence associated with a biological material, comprising: acquiring a first reference image of the biological material; acquiring a first set of one or more images of the biological material using one or more filters corresponding to one or more information dyes; applying the one or more additional dyes to the biological material and then acquiring a second set of one or more images comprising a separate image of the biological material with each of the filters corresponding to the information dyes and a second reference image of the biological material; coregistering the first and second reference images; and then removing any inherent autofluorescence exhibited in the informational images acquired.

Abstract: Methods for detecting a plurality of targets in a biological sample are provided. The method comprises contacting the biological sample with a plurality of target-binding probes to form a plurality of target-bound probes, covalently attaching at least one of the target-bound probes to the biological sample, and observing the signals from the target-bound probes sequentially. An associated kit and device for detection of the plurality of targets are also provided.

Abstract: The invention relates generally to a process of analyzing and visualizing the expression of biomarkers in individual cells wherein the cells are examined to develop patterns of expression by using a grouping algorithm, and a system to perform and display the analysis

Abstract: A method for generating a brightfield type image, which resembles a brightfield staining protocol of a biological sample, using fluorescent images is provided. The steps comprise acquiring two or more fluorescent images of a fixed area on a biological sample, mapping said fluorescent image into a brightfield color space, and generating a bright field image. Also provided is an image analysis system for generating a brightfield type image of a biological sample using fluorescent images.

Abstract: The invention provides a method for antigen retrieval of a formaldehyde-fixed tissue sample comprising incubating a formaldehyde-fixed tissue sample in a first antigen retrieval solution at a temperature of greater than 90° C., transferring the tissue sample to a second antigen retrieval solution, and incubating the tissue sample in the second antigen retrieval solution at a temperature of greater than 90° C. The invention also provides a kit and sample delivery device for carrying out the method.

Abstract: The present application discloses a process for improving the accuracy of fluorescence in-situ hybridization (FISH) assays in which the sample being assayed is likely to contain cells in apoptosis by excluding these cells from the evaluation of the FISH assay. This is conveniently done by labeling the cells in apoptosis by incorporating labeled nucleotides into the apoptosis typical breaks in their nuclear DNA. The present application also discloses a kit and system adapted for carrying out this process for improving the accuracy of FISH assays.

Abstract: Methods for detecting a plurality of targets in a biological sample are provided. The method comprises contacting the biological sample with a plurality of target-binding probes simultaneously to form a plurality of target-bound probes and observing the signals from the target-bound probes sequentially. An associated kit and device for detection of the plurality of targets are also provided.

Abstract: Automated methods and devices that facilitate iterative staining of biological samples from imaging applications are provided. The methods include the steps of providing a small volume flow cell containing a biological sample, applying a stain to the biological sample, combining at least two precursor reagents to form an activated destaining agent and wherein the activated destaining agent decomposition rate is greater than or similar to the destaining reaction rate, and flowing the destaining agent over the biological sample at a flow rate that is greater than the decomposition rate of the activated destaining agent. The process of staining, combining and flowing may be iteratively repeated. Also disclosed herein are devices for iterative staining of biological samples comprising a flow cell, in fluid communication with a premixer, wherein the volume capacity of the premixer is smaller than about five times the volume capacity of the flow cell.

Abstract: The present techniques provide systems and methods for registering images of tissue spots on a tissue microarray (TMA). In studies involving multiple biomarkers being studied on the same TMA, the TMA slide is removed from the microscope, stained, and then imaged, often multiple times. The present techniques relate to validation of the registration of the acquired images of the same TMA. An automatic approach to register the images and detect registration failures as provided herein may enhance the rapid analysis of the tissues. Artifacts such as tissue folding and tissue loss are also determined automatically.

Abstract: The present techniques include methods and systems for finding correspondences between tissue spots in tissue microarray serial sections belonging to the same recipient block. The present techniques may also be used to relate individual tissue cores to clinical information. Using either a whole slide image or the relative x-y coordinates of the tissue spots on the slide, individual tissue spots in different tissue microarrays may be linked to one another and their clinical information.

Abstract: The present techniques provide fully automated methods for quantifying the location, strength and percent of expressed target molecules or other biological markers in immunohistochemically stained biological samples. The samples may be automatically segmented, for example into subcellular compartments, from images of compartmental markers. Then, the distribution of a target molecule on each of these compartments is calculated that includes the percentage and strength of expression. This is different than existing intensity or ratio based methods where abundant low expression levels are indistinguishable from scarce high expression levels.

Abstract: Techniques for removing image autoflourescence from fluorescently stained biological images are provided herein. The techniques utilize non-negative matrix factorization that may constrain mixing coefficients to be non-negative. The probability of convergence to local minima is reduced by using smoothness constraints. The non-negative matrix factorization algorithm provides the advantage of removing both dark current and autofluorescence.

Abstract: Method and systems for removing any inherent autofluorescence associated with a biological material, comprising: acquiring a first reference image of the biological material; acquiring a first set of one or more images of the biological material using one or more filters corresponding to one or more information dyes; applying the one or more additional dyes to the biological material and then acquiring a second set of one or more images comprising a separate image of the biological material with each of the filters corresponding to the information dyes and a second reference image of the biological material; coregistering the first and second reference images; and then removing any inherent autofluorescence exhibited in the informational images acquired.

Abstract: A system for analyzing tissue samples, comprising: a storage device for at least temporarily storing one or more images of one or more cells, wherein at least one of the images is indicative of one or more channels comprising a receptor tyrosine kinase (RTK); and a processing device that determines an extent to which one or more of the RTKs may have translocated from at least one subcellular region to another subcellular region of one or more of the cells; and generates a score based at least in part on the RTK translocation.