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WO2002029710A1 - Method and apparatus for volumetric separation of materials - Google Patents

Method and apparatus for volumetric separation of materials

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Publication number
WO2002029710A1
WO2002029710A1 PCT/US2001/027512 US0127512W WO2002029710A1 WO 2002029710 A1 WO2002029710 A1 WO 2002029710A1 US 0127512 W US0127512 W US 0127512W WO 2002029710 A1 WO2002029710 A1 WO 2002029710A1
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WO
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Prior art keywords
tissue
material
sample
sections
dimensional
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Application number
PCT/US2001/027512
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French (fr)
Inventor
Russell L. Kerschmann
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Resolution Sciences Corporation
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    • GPHYSICS
    • G06COMPUTING; CALCULATING; COUNTING
    • G06TIMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00Image analysis
    • G06T7/10Segmentation; Edge detection
    • G06T7/11Region-based segmentation
    • GPHYSICS
    • G06COMPUTING; CALCULATING; COUNTING
    • G06TIMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00Indexing scheme for image analysis or image enhancement
    • G06T2207/10Image acquisition modality
    • G06T2207/10056Microscopic image
    • GPHYSICS
    • G06COMPUTING; CALCULATING; COUNTING
    • G06TIMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00Indexing scheme for image analysis or image enhancement
    • G06T2207/30Subject of image; Context of image processing
    • G06T2207/30004Biomedical image processing
    • G06T2207/30101Blood vessel; Artery; Vein; Vascular

Abstract

The invention comprises an apparatus for precisely isolating arbitrarily defined volumes of material from samples of tissue (102), manufacturing material, or other substances, and consists of: an imaging device (108) for generating a digital three-dimensional representation of a tissue sample, a repository for tissue sections sequentially removed from a tissue sample (114, 116, 117, 118), computational means (112) for identifying a selected subvolume within the digital three-dimensional representation of a tissue sample and for relating the selected subvolume of the digital representation with subregions of archived tissue sections, and a tissue extraction apparatus capable of identifying, locating and removing a predetermined subregion of a tissue section.

Description

METHOD AND APPARATUS FOR VOLUMETRIC SEPARATION OF MATERIALS

Background of the Invention The light microscope has been traditionally employed in the life sciences for the visualization of biological tissues for the purpose of understanding normal microscopic anatomy and disease processes. Light microscopes are also used extensively in the materials science field to study various manufactured goods. In order to be optimally visualized, the great majority of tissues and some manufactured materials are prepared for microscopy by a process known as histology. In this technique, a sample of the tissue or other material is embedded in a solid medium such as paraffin or plastic and then cut into one or more thin sections on a cutting device called a microtome before being mounted on a glass slide or other support. The sections are often stained with standard or fluorescent dyes to enhance contrast. The sectioning process reveals details in the tissue or material because it greatly reduces the obscuration of one part of the sample by another. Samples may be cut as thin as a few microns, or even a fraction of one micron thick. The staining step not only enhances optical contrast, but also provides information on the chemical composition of the tissue or material through the selective binding by various chemically distinct tissue components to various colored stain constituents.

For more than a century, many millions of histology slides have been observed through standard optical microscopes in biomedical research and clinical anatomic pathology laboratories, and in this time qualitative or semiquantitative analysis has been the predominant use to which microscopes have been applied. More recently, digital microscopes have been introduce, wherein the sectioned sample is electronically imaged through the microscope optics and the resulting digital image is displayed and manipulated on the screen of a computer. Because the image is electronically quantitized, the introduction of digital microscopy has allowed an advance in quantitative analyses of tissue and other materials.

As an alternative to conventional slide-based microscopy, more recently block face microscopy or surface imaging microscopy (as implemented in U.S. Patent 4,960,330, included by way of reference) has become available. In this method, an entire sample is first stained with mixtures of fluorescent stains, and then embedded in an opaque block of plastic or other material. As in standard histology, this block is sectioned on a microtome; but unlike histology it is the resulting freshly cut face of the block rather than the thin section cut from it that is digitally imaged. By manipulating the chemical composition of the stains and the block, a very high-resolution two-dimensional image of the sample contained therein may be obtained. Repeated sectioning and imaging by this method can produce a large number of registered serial sections that can be assembled into a high-fidelity three-dimensional reconstruction of the sample of tissue or material.

In addition to providing a means to visualize microscopic features, new technologies are being created that use the digital microscope as a tool for physical manipulation of tissue for various purposes. In this method, a stained histologically sectioned sample mounted conventionally on a glass slide is positioned under a microscope and a finely focused laser is directed at the sample through the microscope optical path, and aimed by the user to encircle or to scan across selected regions of the tissue section. The laser causes tissue to be burned away or altered in some other way, usually with the goal of isolating some portion of the tissue from the rest of the section. The resulting isolated portion of the tissue is then detached from the slide, either by stripping it away or by applying a sharp pulse of laser light. The isolated sample is then transferred to a suitable vessel for biomolecular analysis. By this method, microscopic samples of tissue and other material of high purity are made available, for example, for gene sequencing, proteomic studies, or other purposes.

Tissue dissection systems work by various methods. One such approach is that of Laser Capture Microdissection (LCM), such as the method developed by Arcturus, Inc., of Mountain Niew, CA, which involves covering the tissue section with a type of thin transparent film that upon laser irradiation becomes adhesive to the portion of the tissue immediately underlying it. Under direct observation, the user exposes only the tissue area to be isolated by scanning the laser beam across it, controlling the beam by means of a joystick or other computer input. The laser light causes the exposed area of tissue to become adherent to the overlying transparent polymer film. The transparent film with the adherent isolated sample is then mechanically stripped from the slide. The tissue is removed from the film in a solvent and thus made available for analysis.

Other systems that have been described include the Laser Pressure Catapulting (LPC) technology, marketed by P.A.L.M. MicroLaser Systems of Bernried, Germany. Instead of heating an area of tissue to be microdissected, the user operating the P.A.L.M. system first focuses the laser into a spot less than a micron in diameter, and circumscribes the area of interest, cutting it free from the surrounding tissue section. Then by applying a sharp pulse of powerful laser energy across the entire area, the system impels the tissue of interest from the glass slide through the force of direct photonic pressure. The P.A.L.M. system thus does not subject the sample of interest to the relatively prolonged high temperatures encountered with the Arcturus system, temperatures which can damage important macromolecules in the sample.

In some implementations of microscope-aided microdissection, instead of directly observing the actual section of tissue, the user works with a digital image of the entire two-dimensional tissue section displayed on a computer screen. The user traces around the area to be isolated, and the computer receives the data from the image program and directs the laser to the corresponding area of the actual tissue section. All current implementations of microscope-aided microdissection involve isolation of a small sample from a single, thin section of tissue or other material. Because standard histologic methods are based on such thin sections that constitute a very small proportion of the total sample, in many cases the amount of material thus isolated is insufficient for certain types of analyses, or to permit multiple analyses from a single sample". In addition, because the manual microtome-based method that produces conventional sections does not permit the precisely directed collection of a particular section through a particular structure of interest within the overall sample, the section that is selected for microdissection may often not contain the widest diameter of the specific material intended for isolation.

There remains a need for a technology with the capability to remove entire three-dimensional volumes of tissue from inside a larger sample. Summary of the Invention In general the invention provides the means for precisely isolating selected volumes of a tissue or other material from larger volumes in order to obtain purified sample volume for chemical analysis and other purposes.

The method includes the steps of (a) generating digital images of planes through a material sample block to obtain a digital three- dimensional representation of the material sample, (b) removing the imaged planes from the material sample block as material sections and archiving the material sections, (c) computationally identifying a selected subvolume within the digital three-dimensional representation of the material sample and relating the selected subvolume of the digital representation with subregions of the archived material sections, and (d) separating the material section subregions from the archived material sections associated with the selected subvolume of the digital representation.

In another aspect of the invention, an apparatus for use in the isolation of a defined volume of material from an actual material sample is provided. The apparatus includes an imaging device for generating a digital three-dimensional representation of a material sample, a repository for physical material sections that have been sequentially removed from a material sample block, computational means for identifying a selected subvolume within the digital three-dimensional representation of a material sample (e.g., a "virtual" material section) and for relating the selected subvolume with the appropriate subregions of the archived material sections, and a material extraction apparatus for removing subregions from the physical material sections identified by the computational means. In yet another aspect of the invention, a means for precisely isolating arbitrarily defined volumes of material from samples of tissue, manufactured material, or other substances, includes 1) means for generating a highly accurate three-dimensional digital representation of samples of whole tissue or other material, 2) means for computationally indicating a defined arbitrary subvolume or subvolumes of tissue or other material within said digital representation, 3) means for producing a highly ordered and precisely positioned series of cut sections comprising the entire sample, 4) means for relating the serial cut sections in an orderly fashion to the said three-dimensional representation, and 5) means for separating said defined arbitrary subvolume of the material in the serial cut sections of the subvolume or subvolumes of interest from the highly registered series of sections of the sample.

In another aspect of the invention, a method for isolating a defined volume of material from a tissue sample, includes removing material from a material sample block and archiving the material sections, generating digital images of the material sections of the material sample block to obtain a digital three-dimensional representation of the material sample; computationally identifying a selected subvolume within the digital three- dimensional representation of the material sample and relating said selected subvolume of the digital representation with subregions of the archived material sections; and separating the material section subregions from the appropriate archived tissue sections. This aspect of the invention recognizes that it may be possible to generate a three dimensional digital representation of the material sample using traditionally cut tissue samples. The digital images may be obtained one at a time, after each material section is cut and archived. Alternatively, all material sections may be cut and archived prior to digital imaging. A wider range of imaging techniques is available to view traditional tissue samples. The material examined in the method and apparatus of the invention may be a biological material, i.e., a materials having origin in an organism or living thing. The material examined in the method and apparatus of the invention may be a manufactured material, i.e., materials acted upon or made by man. It is also within the scope of the invention to examine a natural material, e.g., a mineral or other type of material that occurs in nature. The invention is described herein with reference to a biological material, or tissue; however, it is understood that in all cases a manufactured material or natural material may be similarly used. In preferred embodiments, the planes are sequentially imaged, and preferably sequentially imaging is accomplished by sequentially presenting the surface of the tissue sample block for imaging, or by focusing an optical microscope at sequential greater (or smaller) distances within the tissue sample block. In preferred embodiments, generation of digital images is accomplished using an imaging device selected from the group consisting of a surface imaging microscope, laser scanning confocal microscope and radiologically-based instruments for X-ray computed tomography and magnetic resonance microscopy.

In one preferred embodiment, imaging is complete prior to removing and archiving of the tissue sections. In another preferred embodiment, the imaged plane of the tissue sample block is removed from the tissue sample block immediately subsequent to imaging.

In preferred embodiments, the physical tissue sections are archived by storing the sections at an identifiable position along an surface, which may be an adhesive surface. In other embodiments, the surface may be porous and the sample is attached to the surface using suction drawn through the material.

In other preferred embodiments, the tissue section subregions are removed by microdissection, and preferably by laser microdissection. In other embodiments, microdissection is accomplished using a micromanipulator or by selective ultraviolet irradiation of unwanted areas surrounding said tissue sections subregions.

In preferred embodiments, a selected subvolume is identified by labeling subregions of interest on a consecutive series of two-dimensional images, or by defining a geometric shape within the sample volume. The selected subvolume is related to the corresponding two-dimensional digital images. Alternatively, a subvolume is specified by cell-type or tissue- type. By "archiving" is meant the systematic storage of the physical tissue or material sections removed from the sample block, so that the user is capable of identifying and retrieving any particular tissue or material section. Thus, archiving involves both the storage and identification of the physical tissue or material sections. By "sequentially imaging" is meant that the images are generated in a sequence from one end of the tissue or material sample block to the other. By "sequentially removing imaged planes as tissue or material samples" is meant that physical tissue or material sections corresponding to imaged planes (e.g., "virtual" sections) are serially or consecutively removed, starting from one end of the sample block to the other.

"Tissue section" or "physical tissue section" of "material section" refers to the thin slice of tissue or material that is cut and removed from the tissue sample or material after imaging.

By tissue or material "subvolume" is meant a three-dimensional volume within a three-dimensional digital representation of the volume of an entire tissue or material sample.

By tissue or material "subregion" is meant a two-dimensional region or area of a tissue or material section. The subregion of a tissue section or material section may be identified by direct observation and marking of the two-dimensional digital images or by computational analysis of the selected subvolume.

"Tissue extraction" or "microdissection" refers to techniques used to remove a defined portion or region of interest from a tissue sample. Microdissection is capable of isolating small areas of tissue, as well as single cells. Microdissection may employ various tools, such as micromanipulators for careful removal of selected tissue area. Several commercial methods and apparatuses are available, which rely on laser energy to separate a small, well-defined region of a tissue sample, which is then removed by either adhesion to a polymer film or pressure from a laser light source. Other methods involve the use of ultraviolet radiation for selective destruction of unwanted surrounding material.

Although the invention is particularly useful in the analysis of biological tissues, it is contemplated that the apparatus and method of the invention may be applied to the study of non-biological materials.

Brief Description of the Drawing The invention is described with reference to the figures, which are presented for the purpose of illustration and are not intended to be limiting of the invention, and in which:

Figure 1 is a flow diagram illustrating the steps of the method of the invention;

Figure 2 is an illustration of an apparatus of the invention; and Figure 3 is a pictorial illustration of (A) a strip tape repository of tissue sections indicating the tissue subregions of interest, (B) a computationally generated three-dimensional image of the subvolume of interest related to the tissue subregions of interest, and (C) the preferential extraction of the subregions of interest from the tissues sections. Description of Preferred Embodiments With reference to Figure 1, the method includes digitally imaging sequential planes of a tissue sample block, step 10. In one embodiment, the imaging device generates three-dimensional images by repeatedly cutting away a thin layer of material, e.g., a physical tissue section, from the surface of a block in which a sample is embedded. After each cut, the new surface of the block is imaged to collect a series of two-dimensional digital images that may be reassembled by a computer into a high- resolution three-dimensional replica of the sample. In another embodiment, an optical microscope, e.g., a confocal microscope, may be used to image planes of a tissue sample at varying depths in the sample block. The tissue sections relating to each image may be obtained concurrent with, or subsequent to, imaging. In preferred embodiments, imaging takes place in combination with tissue section removal in a coordinated process.

In coordination with the repeated cutting and imaging of the block face, a series of physical sections, one section for each consecutive digital image, is produced. These physical sections are systematically archived as shown in step 20 for later retrieval and microdissection to remove the tissue of interest. In one embodiment of the invention, the section sample is archived by systematically positioning the physical tissue sections in a repository. The identity (order in removal of the tissue sections from the sample block) and contents (relating to tissue and cell structure of the section) of the tissue sample is related to its location in the repository. The repository may be a plastic strip, and the like, containing identifying information relating to the tissue sections.

Once the sample has been thus sectioned and imaged, the digital volume is visualized on a three-dimensional visualization and analysis software application (step 30). The user manipulates and refines the volume data by examining the graphically displayed three-dimensional tissue features of the exterior of the volume, and by computationally removing portions of the image data in order to examine internal surfaces and other structures. The selection of a suitable subvolume of interest may be carried out in a number of ways. Alternatively, or in concert with three-dimensional examination, the user may arbitrarily select two-dimensional cut-planes through the data volume in order to examine tissue structures in a conventional, two-dimensional histological context. The user may elect to circumscribe structures of interest on a consecutive series of such two- dimensional images, delineating the volume of interest through a series of consecutive contours drawn onto the two-dimensional images (step 40). The images may then be reconstructed into a subvolume of the tissue sample. The three dimensional subvolume may be related to two dimensional physical tissue sections.

Alternatively, a geometric shape may be identified within the sample volume. For example, a sphere, cube, or other suitable subvolume may be defined within the whole tissue volume and the corresponding two-dimensional images (and related physical tissue sections) may be identified.

In more sophisticated applications, it is possible to specify a set subvolume by cell-type or tissue-type. For example, it is possible to specify only that subvolume responding to a selected histological tissue stain. In this manner, it is possible to separate tissues that are chemically, but not morphologically, different.

The result of such manipulations is a delineation, or segmentation, of a set of boundary coordinates in three-dimensional space that define at some suitable level of resolution the volume of tissue the user would like to separate from the tissue sample. These coordinates are transferred to a device that locates the corresponding physical sections of the archived tissue samples, and controls the sequential extraction of the area of interest from each such section. The extracted or dissected tissues are collected in a suitable vial or other vessel for biochemical analysis (step 50). Once this process has been accomplished, a user may wish to return to the sample to harvest some other volume of interest separate from the first volume. As an added feature of the invention, the marked contours delineating any harvested volume of interest may be permanently recorded with the three-dimensional image data, and co-displayed in such a manner as to allow users returning to the three-dimensional image to quickly understand what tissue remains for analysis.

Figure 2 illustrates the general elements of the apparatus of the invention. The apparatus includes a tissue block holder 100 for holding tissue block 102 and a knife blade 104 in holder 106 for removing tissue sections from the tissue block. An imaging device 108 is positioned opposite the exposed surface of the tissue block. The imaging device includes the desired optics and/or laser for probing the sample surface, as well as a video camera for recording the digital images or other device 110, e.g., a computer monitor, for viewing the images. The imaging device is capable of generating digital, highly accurate three-dimensional digital representations of whole tissue or material samples. In a preferred embodiment, the imaging device includes a surface imaging microscope. A surface imaging microscope generates a three-dimensional image by repeatedly cutting away a thin layer of material from the surface of an opaque block in which a fluorochrome- labeled sample is embedded, and imaging the exposed surface.

In other preferred embodiments, other types of microscopes producing digital volumetric information, such as laser scanning confocal microscopes or other optical sectioning technologies, may be used. In confocal microscopy, the sample block is optically transparent, and optical sections at varying depths of field may be sequentially imaged and digitized. The optical sections are imaged in conjunction with serial removal of tissue sections, which may take place during or after imaging. In other embodiments, methods based on radiological techniques such as X-ray computed tomography or magnetic resonance microscopy may be used to produce three-dimensional data. Alternatively, the three- dimensional image can be produced by recording the images of consecutive serial sections of the sample prepared by conventional histology or other means. The tissue block surface may be viewed directly or it may be viewed using a computer-assisted imaging device. For example, the device may be equipped with a video camera or interfaced with a computer monitor for viewing the whole tissue or tissue section images. A wide range of visualization software 112 is commercially available for computationally defining an arbitrary subvolume or subvolumes of tissue or other material within a three-dimensional digital representation. Preferred software applications include the capability to mix and compare two-dimensional tissue section images and three- dimensional tissue volume data. A particularly preferred means for computationally defining an arbitrary subvolume or subvolumes of tissue or other material within a three-dimensional digital representation includes a software application, such as the RESNiew package currently marketed by Resolution Sciences Corporation of Corte Madera, CA, which displays the data volume as a three-dimensional representation with a corresponding two-dimensional cut plane feature, and allows the user to interactively outline arbitrary volumes of the represented tissue sample. Other suitable visualization software may be available from Advanced Visual Systems Inc. of Waltham, MA. By composing a suitable digital export filter and data file format, the defined limits of such data volume can be exported to control the positioning of a tissue extraction apparatus 113, e.g., a laser microdissection apparatus.

In one embodiment of the invention, the section sample is archived by systematically positioning the tissue sections in a repository, such that the identity and content of the tissue sample is related to its location in the repository. For example, referring to Figure 2, the tissue sections may be stored on a continuous strip 114 of polymer such as cinema film or magnetic tape. The strip is fed from take off spool 116, over a receiving surface 117, and onto take up spool 118. The archival device systematically collects physical sections as they are cut from the face of the tissue block and transferred to the continuous strip of polymer film or other material at the receiving surface 117. In preferred embodiments, the strip contains adhesive only in the specific area in contact with the tissue block. Alternatively, the strip may be made up of a porous material and the section may be drawn to the strip using a flow of air directed through the strip.

Figure 3B shows a repository strip 114, including several tissue sections 120 that have been removed and stored on the strip. The tissue sections shown in Figure 3 further include subregions 122 containing thin slices of the subvolume of interest 124 located in the original tissue sample block (Figure 3 A). The tissue section may be catalogued by applying a plastic film with an adhesive surface to the face of the tissue sample before the tissue section is cut from sample block. As the tissue section is cut from the sample block, the plastic tape with the tissue section is lifted from the surface, thereby retaining the tissue section at a known location on the tape. The tissue section position is identified, preferably using a machine readable identifier 126 such as a bar code. In other embodiments, magnetic tape may be used to both secure and identify the tissue sections. A method and device for producing a highly registered, continuous series of physical sections cut from the entire sample is described in U.S. Patent 5,746,855, which is included herewith in its entirety by way of reference. Once the block face has been imaged and the counterpart physical tissue sections have been archived in a suitable repository, the sections can be related in an orderly fashion to the three-dimensional representation of the sample that is obtainable from the visualization software applications described above. The visualization application preferably includes a software database method for indexing each two-dimensional image constituting the three-dimensional data volume to a unique physical section stored on the continuous strip of polymer film. Further, the section serial number or other unique identifier for each section stored on the polymer strip relates the computationally obtained two-dimensional image with a specific tissue section.

Thus, those tissue sections that contain regions of the subvolume of interest are identified and the specific position of the subregion is calculated. Tissue extraction apparatus 113 may then be employed to remove only those regions containing material of interest. In preferred embodiments, the selected subregions are removed by microdissection, for example by use of a micromanipulator-guided needle with an adhesive tip. A preferred method and apparatus for separating the serial sections of the subvolume or subvolumes of interest from the highly registered series of sections of the sample preferably includes a laser capture or laser pressure catapulting method which addresses each physical section in turn and removes only that portion of the tissue residing within the bounds of the arbitrary subvolume or subvolumes of tissue. As each section is processed, the corresponding tissue of interest is collected in a suitable vessel for transfer for biomolecular or other analysis. This selection process is illustrated pictorially in Figure 3C.

Suitable tissue extraction methods include laser capture microdissection (LCM), using the PIXcell II™ LCM system available from Arcturus Engineering (Mountain Niew, CA), and laser pressure catapulting (LPC) using the P.A.L.M. Microlaser available from P.A.L.M. Microlaser Systems (Bernfried, Germany).

Other less preferred embodiments include a technique known as selective ultraviolet radiation fractionation (SURF), which uses a UN- opaque dye to protect the selected areas of the tissue sections against ultraviolet irradiation and obliterates the surrounding unwanted tissue. A similar technique uses an ultraviolet laser beam to obliterate unwanted cells. The remaining material may be collected using various known tools and techniques, such as an adhesive-tipped needle. These techniques suffer from the disadvantage that the surrounding materials are irretrievably destroyed so that subsequent tissue analysis and retrieval are not possible.

The method and apparatus of the invention possesses many advantages for tissue processing and analysis. By providing the user with the ability to work in tissue sample volumes, rather than areas, significantly greater amounts of material are made available with greater efficiency and accuracy. Because of the complex and intertwined nature of biological materials, different cell-types or tissue-types are difficult to separate at a satisfactory level of purity. By using computationally- assisted analysis of the whole tissue sample, in combination with microdissection tissue separation techniques, it is possible to obtain large quantities of pure tissue or cell samples. An additional capability of the invention is the ability to extract quantitative sample amounts, e.g., a known picogram amount of tissue for analysis. It is also contemplated that the method and apparatus of the invention, while well-suited for analysis of biological materials, may be readily used for identification and extraction of other materials, including manufactured materials. For example, it may be useful in the identification of subvolumes within a sample associated with material failure, in order to better understand the nature and/or composition of the material failure.

The invention is described with reference to the following examples, which are presented for the purpose of illustration only, and which are not intended to be limiting of the invention.

Example 1. Extraction of pure sample of blood vessel endothelium.

The study of small blood vessels (microvessels) is a key research topic in cancer research, tissue engineering, and other areas where changes in microvessel densities are important to the development of disease or to the success of artificial tissue implants. Biochemical analysis of endothelial cells has revealed much about how new microvessels are attracted into rapidly growing cancers and what limits their ingrowth into artificial tissues. However, in many cases researchers must depend on cultured endothelial cells for this information, because adequate, pure samples of endothelial cells taken directly from intact tissues cannot be collected by conventional means. Such a pure sample of these cells would represent a more authentic picture of their true biochemical state. Because blood vessels are highly three-dimensional, branched structures, conventional laser capture methods often yield very small amounts of this tissue.

Samples of tumor, cultured tissue, or other material containing microvessels are treated with fluorochrome-conjugated antibodies to specifically label the endothelial tissue with a distinctive fluorescent color, and contrasting color stain is applied to the remainder of the sample. The sample is imaged by surface imaging microscopy to produce a high- resolution three-dimensional representation and simultaneously an ordered series of physical sections are collected. Addressing the three- dimensional image, a researcher uses a visualization and analysis software package to graphically delineate the blood vessels based on their distinctive color, producing a three-dimensional image of the blood vessel network. The three-dimensional coordinates of the limits of this microvascular network are transmitted to a laser microdissection apparatus, which precisely dissects the microvessel cross-sections from each physical serial section. The microdissected tissue is accumulated in a suitable laboratory vial, the embedding polymer is chemically removed, and the purified sample of endothelial tissue is ready for biochemical analysis.

Example 2. Extraction of pure sample of malignant melanoma. Malignant melanoma of the skin may progress in two stages: 1) initially, a relatively slow-growing "superficial spreading" phase will develop, which is sometimes followed by 2) a "vertical growth" phase wherein the growth of the tumor accelerates and invades the deeper layers of the skin. Although its nature is unclear, this transformation is related to a very significant decline in the likelihood of the patient surviving the disease. In order to understand the biomolecular mechanisms behind this transformation, pure samples of superficial spreading and vertical growth phase melanoma would be very desirable.

Samples of melanoma are treated with a standard fluorescent stain that simulates conventional hematoxylin and eosin stain, which allows the pathologist to discriminate between different types of cancer cells. The sample is imaged by surface imaging microscopy to produce a high- resolution three-dimensional representation and simultaneously an ordered series of physical sections are collected. Addressing consecutive two- dimensional cross-sectional images taken through the tissue data volume, a dermatopathologists uses a visualization and analysis software package to graphically delineate the superficial spreading melanoma portion of the cancer. The three-dimensional coordinates of the limits of this tissue subvolume are transmitted to a laser microdissection apparatus, which precisely dissects and separates the tissue subvolume into a vial. The process is repeated for the vertical growth phase portion of the tumor, which is collected in a separate vial. The embedding polymer is chemically removed from each sample, and the purified samples of melanoma tissue are ready for biochemical analysis.

Example 3. Extraction of area of stress failure in a fiber composite material.

Materials scientists study stress failure in certain structural materials, such as carbon fiber composites, in order to produce better and safer materials for aircraft and other applications. In many cases, it would be advantageous to have a purified sample of the material from the region immediately adjacent to the point of failure, in order to produce a better chemical picture of what caused the failure. Since these failures can be microscopic and highly three-dimensional in structure, conventional methods may not produce sufficient amounts of material for analysis. A sample of test material that has been subjected to mechanical stress-induced failure is imaged by surface imaging microscopy to produce a high-resolution three-dimensional representation of the material. Simultaneously an ordered series of physical sections are collected. Addressing the three-dimensional image, the materials scientist uses a visualization and analysis software package to graphically delineate the areas of failure, and the three-dimensional coordinates of the limits of these failure points are transmitted to a laser microdissection apparatus, which precisely dissects the failure cross-sections from each physical serial section. The microdissected material is accumulated in a suitable laboratory vial, the embedding polymer is chemically removed, and the purified sample is ready for chemical analysis. The process may be repeated for subvolumes from areas of the sample that have not shown failure in order to provide controlled comparison.

What is claimed is:

Claims

1. A method for isolating a defined volume of material from a tissue sample, comprising: generating digital images of planes of a material sample block to obtain a digital three-dimensional representation of the material sample; removing said imaged planes from said material sample block as material sections and archiving said material sections; computationally identifying a selected subvolume within said digital three-dimensional representation of the material sample and relating said selected subvolume of the digital representation with subregions of the archived material sections; and separating said material section subregions from the appropriate archived tissue sections.
2. The method of claim 1, wherein the material is a biological material.
3. The method of claim 1, wherein the material is a manufactured material.
4. The method of claim 1, wherein the planes are sequentially imaged.
5. The method of claim 4, wherein the sequentially imaged planes are obtained by sequentially presenting the surface of the material sample block for imaging.
6. The method of claims 4, wherein the sequentially imaged planes are obtained by focusing an optical microscope at sequential distances within the material sample block.
7. The method of claim 1, wherein the imaged plane of the material sample block is removed from the material sample block immediately subsequent to imaging.
8. The method of claim 1, wherein generation of digital images is accomplished using an imaging device is selected from the group consisting of a surface imaging microscope, laser scanning confocal microscope and radiologically-based instruments for X-ray computed tomography and magnetic resonance microscopy.
9. The method of claim 1, wherein said material sections are archived by storing the sections at an identifiable position along an adhesive surface.
10. The method of claim 1, wherein said material section subregions are removed by microdissection.
11. The method of claim 10, wherein said material section subregions are removed by laser microdissection.
12. The method of claim 10, wherein microdissection is accomplished using a micromanipulator.
13. The method of claim 10, said material section subregions are removed by selective ultraviolet irradiation of unwanted areas surrounding said material sections subregions.
14. The method of claim 1, wherein a selected subvolume is identified by identifying subregions of interest on a consecutive series of such two-dimensional images.
15. The method of claim 1, wherein a selected subvolume is identified by a geometric shape within the sample volume, and the selected subvolume is related to the corresponding two-dimensional digital images.
16. The method of claim 1, wherein a selected subvolume is identified by specifying a subvolume by cell-type or tissue-type.
17. A method for isolating a defined volume of material from a tissue sample, comprising: removing material from a material sample block and archiving said material sections; generating digital images of said material sections of said material sample block to obtain a digital three-dimensional representation of the material sample; computationally identifying a selected subvolume within said digital three-dimensional representation of the material sample and relating said selected subvolume of the digital representation with subregions of the archived material sections; and separating said material section subregions from the appropriate archived tissue sections.
18. An apparatus for isolating a defined volume of material from a material sample, comprising: an imaging device for generating a digital three-dimensional representation of a material sample; a repository for material sections sequentially removed from a material sample; computational means for identifying a selected subvolume within said digital three-dimensional representation of a material sample and for relating said selected subvolume of the digital representation with subregions of archived material sections; and a material extraction apparatus capable of identifying, locating and removing a predetermined subregion of a material section.
19. The apparatus of claim 18, wherein the predetermined material section subregion is identified by said computational means sections, whereby a material section subregion is associated with a selected subvolume of the digital representation.
20. The apparatus of claim 18, wherein the imaging device is selected from the group consisting of a surface imaging microscope, laser scanning confocal microscope and radiologically-based instruments for X- ray computed tomography or magnetic resonance microscopy.
21. The apparatus of claim 18, wherein said repository comprises an adhesive strip.
22. The apparatus of claim 21, wherein the adhesive strip contains an identifier for identifying the material section.
23. The apparatus of claim 22, wherein the identifying is computer-readable .
24. An apparatus for isolating a defined volume of material from a material sample, comprising: means for generating a digital three-dimensional representation of a material sample; means for archiving material sections sequentially removed from a material sample; computational means for identifying a selected subvolume within said digital three-dimensional representation of a material sample and for relating said selected subvolume of the digital representation with subregions of archived material sections; and material extraction means for removing a subregion from a material section identified by said computational means.
PCT/US2001/027512 2000-10-02 2001-09-05 Method and apparatus for volumetric separation of materials WO2002029710A1 (en)

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