Transformation of digital images

Image analysis – Applications – Biomedical applications

Reexamination Certificate

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Reexamination Certificate

active

06195451

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to the field of histology.
In present day practice, the preparation of organic tissue samples and other material for transmission microscopy, both visible light and electron microscopy, is normally carried out by subjecting the sample to a series of chemical treatments culminating in the production of a solid block in which the sample is embedded. After the block is produced, thin sections of the sample (with the surrounding embedding material) are cut from the block and transferred to glass slides or other support. The embedding material may then be chemically removed and the tissue section stained with a variety of colored or fluorescent dyes, immunohistochemical stains, or subjected to in situ hybridization prior to examination.
In conventional histopathology, the most common brightfield stain applied to clinically important tissue sections is the hematoxylin and eosin (H&E) formulation. This method results in staining of nucleic acids and other so-called “basophilic” substances in the tissue section with a blue-purple coloration, and proteins and other “acidophilic” or “cosinophilic” tissue components with a pink coloration. This stain is used world-wide as a general screening method for the examination of all tissue components, to be followed in certain cases by special stains that have affinities for specific tissue elements such as microorganisms or nerve processes, and therefore enhance their appearance on stained tissue sections.
Methods have been introduced for en bloc staining, wherein the entire sample is stained by immersion before being subjected to infiltration and embedment. Sections are then cut from the block for transmission microscopy, or the cut face of the block itself is imaged in a process called block face microscopy or surface imaging microscopy. In the latter method, including that implemented in U.S. Pat. No. 4,960,330, a sample that has been stained en bloc with either conjugated or unconjugated fluorescent dyes is subsequently infiltrated by and embedded in a medium, commonly a plastic polymer, that is heavily opacified or otherwise treated to allow for the suppression of images of tissue originating from more than a small number of microns deep within the block. This results in the production of a thin, “virtual section” closely resembling a conventional glass-slide mounted tissue section.
Block face microscopy is advantageous over standard brightfield microscopy in that block face methods allow for the generation of high-quality microscopy images of biological tissue and other materials without the need to manufacture glass histology slides. The elimination of this requirement permits full automation of the histopathologic process, reducing incremental costs for each additional section produced, and consequently allowing for much greater amounts of information to be collected from each sample.
In block face microscopy, the digital virtual section as captured unmodified from the block face is a dark field image resulting from the colored emissions from the fluorescence-stained sample appearing against a black background representing the opacified polymer in which the sample is infiltrated and embedded. In contrast, conventional optical transmission microscopy, including that practiced in most surgical pathology laboratories and other medically-related microscopy-based diagnostic facilities, produces a brightfield image because thin slices of tissue and other material are stained with standard non-fluorescent dyes and are then trans-illuminated with a white or near-white light source, resulting in a background that is brighter, rather than darker than the tissue image.
In order to optimize block face microscopy images for clinical diagnosis and other purposes, it is preferable that the raw darkfield images captured from the face of the block be transformed and displayed as the more familiar images encountered in brightfield microscopy.
SUMMARY OF THE INVENTION
In general, the invention consists of a means for staining a sample with a fluorescent dye, a means for producing a darkfield image of this fluorescent stained sample, and a means of transforming the darkfield images into bright field images for examination on a computer monitor. The process includes applying a digital lookup table or other computational means in order to convert the darkfield data to brightfield forms, and a means of displaying the transformed information. Preferably, the imaging means is a block face microscope, and the means for transforming the images is a digital computer.
Accordingly, in a first aspect, the invention features an image production method that includes: (a) staining a sample with a fluorescent dye; (b) producing a first image of the resultant fluorescent-dyed sample; and (c) using a digital processor to convert the first image to a second image that mimics an image of the sample stained with a non-fluorescent dye.
In a preferred embodiment, the converting of the first image to the second image includes applying a lookup table to the first image. In preferred embodiments, the modifying includes inverting the lookup table, or adjusting the color ranges of either the first image or the second image to mimic the color ranges of the sample stained with a non-fluorescent dye.
The sample can be stained with one dye or with two or more dyes. A preferred sample is a biological sample.
The invention also features an image production method that includes: (a) staining a sample with a first and a second fluorescent dye; (b) producing a first image of the sample stained with the first dye and a second image of the sample stained with the second dye; and (c) using a digital processor to convert the first and second images to a third image that mimics an image of the sample stained with hematoxylin and cosin.
The invention features an image production method that includes: (a) staining a sample with a fluorescent dye; (b) producing a first image of the sample stained with the dye and a second image of the sample stained with the dye; and (c) using a digital processor to convert the first and second images to a third image that mimics an image of the sample stained with hematoxylin and cosin.
The invention also features an image production method that includes: (a) staining a sample with the plurality of fluorescent dyes; (b) producing a first image of one of the dyes in the sample; and (c) using a digital processor to convert the first image to a second image that mimics an image of the sample stained with one non-fluorescent dye.
In another aspect, the invention features an image production method that includes: (a) staining the sample with the plurality of fluorescent dyes; (b) producing a first image that mimics the sample stained with a subset of fluorescent dyes; and (c) using a digital processor to convert the first image to a second image that mimics an image of the sample stained with one or more non-fluorescenit dyes.
Preferably, the first and second images of the invention are produced using an apparatus that includes a block-face microscope. The dye used in the invention can include a metachromatic dye (e.g., acridine orange).
By “conjugated” dye is meant a dye that is bound to a molecule having specificity for tissue elements. Exemplary conjugated dyes include, without limitation, fluorescently-coupled antibodies and fluoresc dna probes.
By “unconjugated” dye is meant a dye that is inherently fluorescent.
By “infiltration” is meant treating the tissue with a liquid or series of liquids which penetrate throughout the tissue to the molecular level and are then transformed into a solid in order to render the sample rigid.
By “embedding” or “embedment” is meant positioning the infiltrated tissue in a mold and surrounding it with a substance (usually the same as the infiltrating substance) which is then hardened to form an encasing block. The embedding substance thus serves to provide rigid support and to facilitate the cutting process.
By “sectioning” is meant cutting from the block thin slices which may then be

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