Abstract: Approaches are disclosed for calibrating a plurality of imaging devices, such as microscopes. In certain implementations, a calibration plate is employed that includes a variety of calibration features. The calibration features comprise a geometric calibration, an illumination calibration, and an optical calibration. Imaging devices calibrated in accordance with the present approaches may be used to generate images having consistent attributes, such as brightness, regardless of which imaging device is employed.

Abstract: Referencing of image acquired in multiple rounds of imaging is disclosed. In certain implementations, a baseline round of images are acquired and registered to one another to establish a global transformation matrix. In a subsequent round of image acquisition, a limited number of field of view images are initially acquired and registered to the corresponding baseline images to solve for translation, rotation, and scale. The full set of baseline images is then acquired for the subsequent round and each image is pre-rotated and pre-scaled based on the transform determined for the subset of images. The pre-rotated, pre-scaled images are then registered using a translation-only transform.

Abstract: Methods comprising probing multiple targets in an H&E stained biological sample are provided. The methods include the steps of providing a hematoxylin and eosin stained biological sample containing multiple targets, optionally detecting H&E staining of the sample, removing the hematoxylin and eosin signals, and detecting additional features or targets in the biological sample. The detecting step may include performing the steps of binding at least one probe to one or more targets to the sample, detecting a signal from the probe and contacting the sample with a bleaching agent. The process of binding, detecting and bleaching may be iteratively repeated.

Abstract: Approaches are disclosed for calibrating a plurality of imaging devices, such as microscopes. In certain implementations, a calibration plate is employed that include a variety of calibration features. Imaging devices calibrated in accordance with the present approaches may be used to generate images having consistent attributes, such as brightness, regardless of which imaging device is employed.

Abstract: The invention provides a slide holder that utilizes edge detection algorithms to determine the location of various edges of a slide. From these edges, and integrating characteristic slide dimension, both linear offset of the edge of the slide in the holder and any rotation of the slide relative to the microscope stage axis are determined. The process of using the slide holder of the invention ensures precise and accurate placement of a slide during a first round of imaging and for reload in subsequent rounds.

Abstract: Approaches are disclosed for calibrating a plurality of imaging devices, such as microscopes. In certain implementations, a calibration plate is employed that includes a variety of calibration features. The calibration features comprise a geometric calibration, an illumination calibration, and an optical calibration. Imaging devices calibrated in accordance with the present approaches may be used to generate images having consistent attributes, such as brightness, regardless of which imaging device is employed.

Abstract: The invention provides a slide holder that utilizes edge detection algorithms to determine the location of various edges of a slide. From these edges, and integrating characteristic slide dimension, both linear offset of the edge of the slide in the holder and any rotation of the slide relative to the microscope stage axis are determined. The process of using the slide holder of the invention ensures precise and accurate placement of a slide during a first round of imaging and for reload in subsequent rounds.

Abstract: Methods comprising probing multiple targets in an H&E stained biological sample are provided. The methods include the steps of providing a hematoxylin and eosin stained biological sample containing multiple targets, optionally detecting H&E staining of the sample, removing the hematoxylin and eosin signals, and detecting additional features or targets in the biological sample. The detecting step may include performing the steps of binding at least one probe to one or more targets to the sample, detecting a signal from the probe and contacting the sample with a bleaching agent. The process of binding, detecting and bleaching may be iteratively repeated.

Abstract: Approaches are disclosed for calibrating a plurality of imaging devices, such as microscopes. In certain implementations, a calibration plate is employed that include a variety of calibration features. Imaging devices calibrated in accordance with the present approaches may be used to generate images having consistent attributes, such as brightness, regardless of which imaging device is employed.

Abstract: Methods comprising the use probing multiple targets in a H&E stained biological sample are provided. The methods include the steps of providing a hematoxylin and eosin stained biological sample containing multiple targets, observing the sample, removing the hematoxylin and partially removing the eosin by washing the sample, contacting the sample with a borate salt, and irradiating the sample to remove the residual eosin fluorescence. The method further includes the optionally performing the additional steps of binding at least one probe to one or more targets to the sample, observing a signal from the probe and contacting the sample with a bleaching agent. The process of binding, observing and bleaching may be iteratively repeated.

Abstract: A method of analyzing a biological sample suspected of having classic Hodgkin lymphoma, comprising (1) detecting, in a single sample, for the expression of at least two biomarkers selected from CD30, CD15, CD20, CD45, CD3, Pax-5, CD79A, BOB1 and OCT-2; and (b) analyzing the sample based on the presence, absence and/or expression level of the at least two biomarkers. Also provided is a method wherein all the nine biomarkers are analyzed on a single sample. Further provided are method for diagnosing classical Hodgkin lymphoma, as well as system and kit that comprise the means for executing the novel methods.

Abstract: Automated assessment of registration quality, focus, and area defects in sequentially acquired images, such as images acquired by a digital microscope, is disclosed. In one embodiment, acquired images are registered and whole-image defects are automatically detected based on a figure of merit generated by the registration process. In related implementations, area defects may be automatically detected by calculating correlations in localized image regions for images acquired in different imaging rounds.

Abstract: A method for providing a composite image of a single biological sample from a patient suspected of having classical Hodgkin lymphoma, comprising the steps of generating a first image of the biological sample including the presence, absence and/or expression level of a first biomarker, generating a second image of the biological sample including the presence, absence and/or expression level of a second biomarker, and generating a composite image that provides the relative location or expression of both biomarkers. Also provided is a method of analyzing a biological sample, comprising providing a composite image of the biological sample according to the method for providing a composite image, and analyzing the expression of the biomarkers of interest from the composite image. Further provided are method for diagnosing classical Hodgkin lymphoma, as well as system and kit that comprise the means for executing the novel methods.

Abstract: In an imaging device having an objective and a stage for holding a sample to be imaged, a method for autofocusing is presented. The method includes determining a measured focus value corresponding to at least a first of a plurality of logical image segments. Further, the method includes imaging the first logical image segment using the measured focus value. The method also includes determining a predicted focus value for a second of the plurality of logical image segments using the measured focus value and a stored focus variation parameter. In addition, the method includes imaging the second logical image segment using the predicted focus value.

Abstract: Referencing of image acquired in multiple rounds of imaging is disclosed. In certain implementations, a baseline round of images are acquired and registered to one another to establish a global transformation matrix. In a subsequent round of image acquisition, a limited number of field of view images are initially acquired and registered to the corresponding baseline images to solve for translation, rotation, and scale. The full set of baseline images is then acquired for the subsequent round and each image is pre-rotated and pre-scaled based on the transform determined for the subset of images. The pre-rotated, pre-scaled images are then registered using a translation-only transform.

Abstract: Automated assessment of registration quality, focus, and area defects in sequentially acquired images, such as images acquired by a digital microscope, is disclosed. In one embodiment, acquired images are registered and whole-image defects are automatically detected based on a figure of merit generated by the registration process. In related implementations, area defects may be automatically detected by calculating correlations in localized image regions for images acquired in different imaging rounds.

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, generating a brightfield image, and optionally applying a sharpening transformation correction. Also provided is an image analysis system for generating a brightfield type image of a biological sample using fluorescent images.

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, generating a brightfield image, and optionally applying a sharpening transformation correction. Also provided is an image analysis system for generating a brightfield type image of a biological sample using fluorescent images.

Abstract: A method for imaging is presented. The method includes acquiring a plurality of images corresponding to at least one field of view using an imaging device having an objective and a stage for holding a sample to be imaged. Furthermore, the method includes determining a figure of merit corresponding to each pixel in each of the plurality of acquired images, wherein the figure of merit comprises a discrete approximation to a gradient vector. The method also includes synthesizing a composite image based upon the determined figures of merit.

Abstract: A method of autofocusing includes capturing first, second and third images of a sample, at respective first, second and third sample distances and respective first, second and third lateral positions determined with respect to an objective; determining a quantitative characteristic for the first, second and third images; determining a primary sample distance based upon at least the quantitative characteristics for the first, second, and third images; and capturing a primary image of the sample at the primary sample distance and at a primary lateral position that is offset from the first, second and third lateral positions.