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EP1216408A1 - Systeme de traitement et de mise en image d'echantillons - Google Patents

Systeme de traitement et de mise en image d'echantillons

Info

Publication number
EP1216408A1
EP1216408A1 EP00952641A EP00952641A EP1216408A1 EP 1216408 A1 EP1216408 A1 EP 1216408A1 EP 00952641 A EP00952641 A EP 00952641A EP 00952641 A EP00952641 A EP 00952641A EP 1216408 A1 EP1216408 A1 EP 1216408A1
Authority
EP
European Patent Office
Prior art keywords
sample
block
container
imaging
tissue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00952641A
Other languages
German (de)
English (en)
Inventor
Russell L. Kerschmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Resolution Sciences Corp
Original Assignee
Resolution Sciences Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Resolution Sciences Corp filed Critical Resolution Sciences Corp
Publication of EP1216408A1 publication Critical patent/EP1216408A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts

Definitions

  • the invention relates to the field of histology.
  • tissue samples and other materials for transmission microscopy are 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.
  • the tissue sample is first chemically fixed with formalin, glutaraldehyde, or other material which serves to preserve the sample from autolysis (self-degradation), to render the sample rigid, and to increase its permeability, thereby enhancing the infiltration of the subsequent solutions.
  • the infiltration steps which follow chemical fixing remove all of the water from the sample through progressive replacement of water with increasing concentrations of solvents such as alcohol and xylene.
  • Infiltration is followed, for example, by treatment with melted paraffin, and the sample then is cooled to room temperature whereupon it solidifies.
  • the tissue is infiltrated with plastic polymer that is then hardened by heat, ultraviolet light or other means. The hardened, infiltrated tissue is then positioned in a mold and surrounded with paraffin or plastic to produce a tissue block.
  • the present invention allows for the consolidation and simplification of the processing and imaging of tissue samples.
  • clinicians and scientists the clients send tissue samples to a processing center and later receive hardcopy or digital images of the sectioned samples.
  • the client can modify the digital images, or even produce new digital images from the data (i.e., the sum of digital images) provided by the processing center.
  • the invention features a system for processing a sample, including: (a) a container for storing and transporting the sample; (b) means for infiltrating and embedding the sample to form a block in the container; (c) means for sectioning the sample in the block; (d) means for imaging the sample in the block; and (e) means for identifying the sample during its processing.
  • the system of the invention can include means for producing and storing a digital image of the sample, as well as a computer program for visualizing the digital image.
  • the system can also include means for chemically fixing the sample prior to infiltrating and embedding.
  • the invention features a method of imaging a sample. The method includes: (a) providing a container suitable to hold a sample; (b) chemically fixing the sample in the container; (c) infiltrating and embedding the sample to form a block in the container; (d) sectioning the sample in the block; and
  • the method includes step (f), storing the image as a digital image, as well as step (g), visualizing the digital image.
  • sample tissue or other material which is to be stained and embedded.
  • the thickness of a sample can be greater than 200 microns, one millimeter, one centimeter, or more.
  • fixation is meant the treatment of tissue specimens with a chemical solution that preserves, hardens, and permeabilizes the sample.
  • 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.
  • 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.
  • sectioning is meant cutting from the block thin slices.
  • staining is meant treating a material with a colored or fluorescent substance that associates with the material on the molecular level.
  • Fluorescence or darkfield staining is accomplished using unconjugated dyes (i.e., dyes that are naturally fluorescent when excited with light of the proper wavelength) or conjugated dyes (i.e., molecules that bind to the sample and that are attached, either directly or indirectly, to a fluorochrome).
  • conjugated dyes include without limitation: (i) a primary antibody that binds to an antigen and a secondary antibody, containing a fluorochrome, that binds to the primary antibody; and (ii) a molecule that has, covalently bound to it, a fluorochrome.
  • Fluorescence staining results in images that have a black background, while "standard” or “brightfield” staining is accomplished using dyes that are usually non-fluorescent and results in images with a bright, or white background. It is understood that the same dye could be useful for both fluorescence and standard microscopy.
  • the invention provides a method for the accumulation of more data (in the form of digital images) without an increase in material and labor costs.
  • the system of the present invention includes the following general components: a container for storing and transporting the sample; means for infiltrating and embedding the sample to form a block in the container; means for sectioning the sample in the block; means for imaging the sample in the block; and means for identifying the sample during the processing of the sample.
  • the system also includes means for chemically fixing the sample prior to infiltrating and embedding, although this is not essential.
  • the system also includes: means for producing and storing a digital image of the sample; and a computer program for visualizing the digital image.
  • 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. Additionally, the images are readily stored in digital format, facilitating computer aided analysis of the images, such as measurement of area or volume, or three dimensional reconstruction.
  • the digital "virtual" section 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.
  • 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.
  • the raw darkfield images captured from the face of the block can be transformed and displayed as the more familiar images encountered in brightfield microscopy. Methods for such image transformation are described, for example, in U.S.S.N. 09/31 1,789, which is hereby incorporated by reference.
  • the container The tissue sample of interest is placed in a container that, preferably, is compatible with all steps of tissue processing, including storing, transporting, chemically fixing, infiltrating, embedding, sectioning, and imaging.
  • the container preferably serves as a mold for use in the preparation of a sample to be sectioned and examined.
  • the mold will define the shape of the block in which the tissue sample is embedded.
  • the mold defines an elongated cavity, including an opening at a proximate end for introduction of a tissue sample from outside the mold. The proximate end defines a shape adapted for engagement with a securing means.
  • the mold tapers to a distal end which defines a polyhedron of a size and shape suitable for sectioning; this polyhedron is referred to as the specimen chamber.
  • the shape of the mold should be compatible with the size and shape of both the imaging field and the tissue sample.
  • the distal end of the mold preferably approximates the size and shape of an imaging field of an imaging apparatus which may be used to view an emerging face of a tissue block prepared from the mold, such that the cross-section of the specimen chamber transverse to the length of the block, or the exposed surface of the sample chamber, approximates or is of similar size and shape as the imaging field of an imaging apparatus used to view the surface.
  • the imaging field is the area over which an imaging device can record data which relates to the surface.
  • the imaging device provides microscopic imaging of the surface, that is, the actual scale of the imaged area is not dictated by the scanning array but rather by the magnification optics.
  • the size of the imaging field is determined by the magnification optics and the shape of the imaging field is dictated by the array design.
  • a suitable container shape is selected based on tissue sample size and optimal imaging geometry. For example, a punch biopsy of the skin may produce a round skin sample of about 4 mm in diameter.
  • a container having a 4.5 mm x 4.5 mm square specimen chamber readily accommodates such a sample and, results in a block with an imaging corresponding substantially to the imaging field.
  • the shape of the specimen chamber may be selected accordingly.
  • a prostate needle biopsy produces a sample that is approximately 1 mm in diameter and 17 mm in length.
  • a preferred container for such a tissue sample would possess a specimen chamber having that shape as well, for example, by having a rectangular cross-section with dimensions of about 2 mm across and 20 mm long.
  • a linear CCD scanner may be used to generate a rectangular imaging field.
  • the quality of a section cut from such a long thin block face may be less than optimal for examination with conventional microscopy techniques, the quality of the exposed, cut face is unaffected.
  • a wide variety of shapes of the container not heretofore considered suitable for tissue imaging, can be employed in order to obtain high quality imaging area.
  • the container may be prepared from any conventional material, for example, polypropylene, silicone or polystyrene. Selection of the material may be influenced, in part, by the intended use of the container.
  • the container may be reusable or sacrificial. Reusable containers are prepared from flexible materials such as silicone.
  • Sacrificial containers include containers which remain on the molded block during sectioning operations, as well as containers that are destructively removed from the molded block.
  • the sacrificial containers desirably are prepared using materials that fracture or tear easily. Suitable materials include polypropylene.
  • the container desirably is prepared from rigid or sturdy material that lends itself to being cut. Suitable materials include polystyrene.
  • the container is threaded so that a cap may be used to close the container.
  • a cap may be used to close the container.
  • snap-on caps or other leak-proof caps may be used.
  • the container is preferably labeled so that it can be distinctly identified. There are many means by which a container can be identified as being distinct from all other containers; any of these means can be used in the present invention. In one example, each container is labeled with a unique bar code. Such labeling of containers allows for the identification of a sample during processing of large batches of samples.
  • a tissue sample is embedded into a molded block using standard embedding methods.
  • the tissue is first chemically fixed with standard fixative liquids to preserve the sample from autolysis (self-degradation), to render the sample rigid, and to increase its permeability, thereby enhancing the infiltration of the subsequent solutions.
  • the water from the sample is removed through progressive replacement of water with increasing concentrations of solvents such as alcohol and xylene.
  • Infiltration is followed by treatment with melted paraffin or with plastic polymer to produce a hardened infiltrated tissue.
  • the preceding steps are carried out in the container.
  • the hardened, infiltrated tissue is then placed in the specimen chamber of the container and is surrounded with paraffin or plastic to produce a tissue block.
  • a sectioning apparatus typically includes a reciprocating bar supporting a tissue block holder and a knife holder with a blade.
  • the reciprocating bar moves the tissue holder (and the molded block secured thereto) up and down and advances the molded block towards the blade to remove a section of the block.
  • the tissue sections cut from the block are discarded or are saved for later use, as described in U.S. Patent No. 5,746,855, hereby incorporated by reference, and the exposed face of the block is examined.
  • the microtome is positioned on a base which also supports an imaging apparatus for viewing the exposed face of the block.
  • Typical imaging apparatuses which can be used in the imaging of the molded block of the invention include area array and linear charge coupled devices (CCDs).
  • a CCD is a detector that provides digital images. The digital format allows the images to be manipulated by a computer, which can electronically modify or copy them. Electronic storage and editing of images provide significant advantages over conventional photographic methods.
  • the viewing field of an area array scanner is a rectilinear area defined by a two-dimensional array of pixels. The greater the number of pixels defining the image, the greater the resolution.
  • a low resolution area array CCD may have dimensions of 300 x 600 pixels, while higher resolution CCDs have dimensions on the order of 2000 x 2000 pixels.
  • a linear CCD scanner has only one or a few rows of pixels. A complete image is obtained by incrementally advancing the linear CCD (or the sample) as the scanner moves across the width of the sample. In both case, the imaging field defines a rectilinear area. Rarely, non-rectilinear CCDs may be employed.
  • the selection of the size and shape of the specimen chamber is made with consideration of the type of imaging apparatus used to capture the images from the exposed face of the block.
  • the specimen chamber shape is selected to avoid empty space in the imaging area or extension of the sample outside the imaging area. These circumstances are to be avoided where possible because they reduce the amount of digital data (pixels) that correspond to the tissue sample.
  • imaging resolution of the sample is improved.
  • the acquired image can be stored in digital format using any available storage medium.
  • a preferred medium is one that can be easily transported, for example by mail, or one that can be sent to the client electronically (e.g., by the internet).
  • the system of the invention can be used to process any type of sample, whether biological or non-biological.
  • biological samples are biopsy and autopsy samples.
  • non-biological samples are samples from the paper industry (e.g., paper towels), mineral samples from the mining industry, and polymer and plastic samples used in manufacturing (e.g., of automobiles).
  • the client collects a punch biopsy of skin from a patient or animal and places it in a container, labeled with a bar code and containing chemical fixative.
  • the container and the fixative are each provided by the processor (the person or company that sections and images the sample).
  • the processor also supplies the chemical fixative, which may be supplied in the container or, alternatively, in a second container (and, thus, transferred to the first container by the client).
  • the client places a threaded cap onto the container.
  • the client records the bar code number and the sample contents for later identification, and then sends the sample by appropriate means to the processor.
  • the processor scans the bar code into the computer that will collect the digital images of the sectioned sample.
  • the container is placed in an automated sample processing machine (e.g., Leica LYNX auto tissue processor; Wetzlar, Germany), and the sample is processed through a series of processing solutions (e.g., stains, graded alcohols, and/or xylene or other organic solvent), then liquid polymer containing optical additives (e.g., an opacifier).
  • an automated sample processing machine e.g., Leica LYNX auto tissue processor; Wetzlar, Germany
  • processing solutions e.g., stains, graded alcohols, and/or xylene or other organic solvent
  • liquid polymer containing optical additives e.g., an opacifier
  • the sample Prior to hardening of the polymer into a solid block, the sample is placed in the specimen chamber at the tip of the container. This can be easily achieved, for example, by centrifugation. The orientation of the sample relative to the sectioning plane is unimportant, because orientation of the digital image can be performed by a computer during visualization.
  • the polymer is hardened using any appropriate method (e.g., heat, UV, time, etc.) Once hardened, the sample and the block are mounted on a microtome for sectioning. In one embodiment, the block is left in the container, and the container is sectioned along with the embedded sample. Alternatively, the container can be removed, either entirely or partially (e.g, only the container adjacent the tissue chamber), prior to sectioning. The sample is then sectioned and imaged.
  • Digital images of the sample are stored in a computer. Following sectioning, the block can be discarded or, if unsectioned sample remains in the block, stored for later use. The sections themselves can also be discarded or saved (e.g., for archival purposes).
  • One method for collecting sections is described in U.S. Patent No. 5,746,855.
  • the sections, or images of the sample be labeled with a bar code number corresponding to the one on the sample container.
  • the digital images, appropriately labeled, are sent to the client using a suitable storage medium (e.g., diskette, CD, and the like).
  • the digital images are transferred to the client electronically (e.g., by the internet). It may be desirable to compress the computer file for greater ease in sending the digital image. It is understood that the format of the digital images is unimportant provided the client has the means to view the images.
  • the processor provide to the client computer software for viewing of the images. Such software may also allow further modifications (e.g, three-dimensional reconstruction, imaging of the XZ or YZ plane, etc.).
  • the system of the present invention may be used in conjunction with any conventional microscopic tissue preparation techniques.
  • the invention may be used in conjunction with the stains and embedding polymers described in co- pending application U.S.S.N. 09/154,430, hereby incorporated in its entirety by reference.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Multimedia (AREA)
  • Theoretical Computer Science (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Cette invention concerne un système de traitement d'échantillons qui comprend: (a) un récipient destiné au stockage et au transport d'un échantillon; (b) un dispositif permettant d'infiltrer et d'enrober ledit échantillon sous forme de bloc dans le récipient; (c) un dispositif permettant de sectionner l'échantillon dans le bloc; (d) un dispositif de mise en image de l'échantillon dans le bloc ; et (e) un dispositif d'identification de l'échantillon pendant son traitement.
EP00952641A 1999-08-25 2000-08-08 Systeme de traitement et de mise en image d'echantillons Withdrawn EP1216408A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US38308599A 1999-08-25 1999-08-25
US383085 1999-08-25
PCT/US2000/021627 WO2001014853A1 (fr) 1999-08-25 2000-08-08 Systeme de traitement et de mise en image d'echantillons

Publications (1)

Publication Number Publication Date
EP1216408A1 true EP1216408A1 (fr) 2002-06-26

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP00952641A Withdrawn EP1216408A1 (fr) 1999-08-25 2000-08-08 Systeme de traitement et de mise en image d'echantillons

Country Status (6)

Country Link
EP (1) EP1216408A1 (fr)
JP (1) JP2003507734A (fr)
CN (1) CN1372638A (fr)
AU (1) AU6530100A (fr)
TW (1) TW463042B (fr)
WO (1) WO2001014853A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7372985B2 (en) 2003-08-15 2008-05-13 Massachusetts Institute Of Technology Systems and methods for volumetric tissue scanning microscopy
US20110224574A1 (en) * 2008-11-13 2011-09-15 Sadler John W Methods and systems for tissue processing and imaging

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4960330A (en) * 1985-07-16 1990-10-02 Kerschmann Russell L Image recording apparatus
US5991028A (en) * 1991-02-22 1999-11-23 Applied Spectral Imaging Ltd. Spectral bio-imaging methods for cell classification

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0114853A1 *

Also Published As

Publication number Publication date
WO2001014853A1 (fr) 2001-03-01
JP2003507734A (ja) 2003-02-25
TW463042B (en) 2001-11-11
AU6530100A (en) 2001-03-19
CN1372638A (zh) 2002-10-02

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