The United States of America, As Represented by the Secretary, Department of Health and Human Services

The United States of America As Represented by the Department of Health

The United States of America As Represented by the Secretary of

The United States of America

John I Peterson

Seth R Goldstein

Paul D Smith

Robert F Bonner

Lance A Liotta

David B Krizman

Michael R Emmert-Buck

Thomas J Pohida

W Marston Linehan

Rodrigo F Chuaqui

Jeffry M Trent

Harold W Tipton

Tristan Gorrindo

Raphael V Fitzgerald

A method of microdissection which involves forming an image field of cells of the tissue sample utilizing a microscope, identifying at least one zone of cells of interest from the image field of cells which at least one zone of cells of interest includes different types of cells than adjacent zones of cells, and extracting the at least one zone of cells of interest from the tissue sample. The extraction is achieved by contacting the tissue sample with a transfer surface that can be selectively activated so that regions thereof adhere to the zone of cells of interest to be extracted. The transfer surface includes a selectively activatable adhesive layer which provides, for example, chemical or electrostatic adherence to the selected regions of the tissue sample. After the transfer surface is activated, the transfer surface and tissue sample are separated. During separation, the zone of cells of interest remains adhered to the transfer surface and is thus separated from the tissue sample, the zone of cells of interest may then be molecularly analyzed.

Isolation of cellular material under microscopic visualization

A tissue sample is conventionally visualized in a microscope. A selectively activated convex surface is provided, preferably at the distal end of a rod. This selectively activated convex surface when activated, typically with a laser through an optic light path in the microscope, provides the activated region with adhesive properties. At least one portion of the tissue sample which is to be extracted is identified. This identified portion is contacted with a portion of the selectively activated convex surface on the end of the rod. When the convex surface is activated, typically by exposure to laser light in the footprint of the desired sample, an adhesive transfer surface on the selectively activated convex surface is provided which adheres to the desired cells in the footprint of the desired sample. Thereafter, the adhesive transfer surface is separated from the remainder of the tissue sample while maintaining adhesion with the desired cells. Thus the desired portion of the tissue sample is extracted. The disclosed selectively activated convex surface is preferably utilized to collect desired tissue samples at more than one location on the same slide or from different slides. The collected tissue samples can thereafter be inspected if desired, as collected on the convex surface, and then liberated&mdash;as by dissolving the proteins of the samples. This can effectively concentrate rarely occurring cells in order to obtain enough pure material for analysis. A rod having a convex surface with the selectively activated material is set forth as a staple for use with the apparatus and process. Preferred shapes for the convex surface are disclosed as well as a method for coating rods with a resultant rod article.

Convex geometry adhesive film system for laser capture microdissection

A method and apparatus of gathering by LCM identified cellular material from randorn locations on a tissue sample to designated locations on a transporting substrate enables convenient further processing. A transporting substrate has an identified mapped location for receiving identified cellular material. At least a segment of a selectively activatable coating is placed on the side of the transporting substrate in apposition to the tissue sample at the mapped location. The transporting substrate and sample are relatively moved to place the selectively activated coating at the mapped location in apposition to identified cellular material of the tissue sample which is to be extracted. Thereafter, the selectively activatable coating is activated and impressed or impressed and activated to form an adhesive region on the transporting substrate for adhering to the identified cellular material. Upon removal of the transporting substrate from the tissue sample, identified cellular material adheres to the transporting substrate at the mapped location.

Mechanical handling systems for laser capture microdissection

A low temperature melt film such as EVA is prepared for laser capture microdissection by having a thin specimen non-adhering coating in the range of 0.1% to 10% of the total film thickness placed on the sample exposed side of the film. When the film is brought into contact with the specimen, the specimen non-adhering coating prevents non-specific transfer due to sticky adherence of portions of the sample. At the same time, the non-adhering coating on the low temperature melt film surface can stabilize and protect the low temperature melt film against variations in performance due to ambient humidity and temperature variation. Upon appropriate heating for laser capture microdissection, the barrier of the thin coating allows conventional film melting with otherwise uninhibited adhesion of selected cell areas to the film. Coatings on the low temperature melt film (EVA) surface in selected locations is made by applying film-forming material from a volatile solvent-based solution, followed by evaporation of the solvent. The coating solution can be applied by spraying, dipping, or adding exact volumes to a surface with a micropipet. Spreading a measured volume of solution over the surfaces can coat flat surfaces. The coating thickness can be controlled by the volume and concentration of coating solids added to a known area.

Surface coatings for hot-melt adhesive film

A process of microdissection where a tissue sample is conventionally visualized in a microscope. A selectively activatable convex surface is provided, preferably on the periphery at the distal end of a rod. This selectively activatable convex surface when locally activated, typically with a laser through an optic light path in the microscope, provides the activated region with adhesive properties. The tissue sample has at least one portion, which is to be extracted is identified. This identified portion is contacted with a portion of the selectively activatable convex surface on the periphery of the rod. When the convex surface is locally activated, typically by exposure to laser light in the footprint of the desired portion, an adhesive transfer surface on the selectively activatable convex surface is activated which adheres to the desired cells in the footprint of the desired portion. Thereafter, the adhesive transfer surface is separated from the remainder of the tissue sample while maintaining adhesion with the portion of the sample. Thus the desired portion of the tissue sample is extracted. The disclosed selectively activatable convex surface is preferably utilized to collect desired tissue samples at more than one location on the same slide or from different slides. A rod having a convex surface with the selectively activatable material is set forth as a staple for use with the apparatus and process. Preferred shapes for the convex surface are disclosed as well as a method for coating rods.

Method utilizing convex geometry for laser capture microdissection

A method for correcting common path variations in intensity in fiber optic chemical sensing devices uses a device for spatially separating light of different wavelength regions and a dye system selected so that light passing back to a measuring system along the fiber optic sensor consists of two wavelength regions. The first wavelength region varies with the concentration of analyte, and the other wavelength region is insensitive to the concentration of analyte.

Method for obtaining a ratio measurement for correcting common path

Plastic optical fibers are joined by heat-flaring their ends in a first tubular sleeve and joining the flared ends within a tubular sleeve of about the same size with a U.V.-curable optical cement. The fibers may be joined side-by-side or end-to-end. Different diameter tubes may also be joined together by the method of the present invention.

Method of joining plastic optical fibers and connections obtained

A fiber optic probe to be implanted in human body tissue for physiologic studies involving measurement and monitoring of the partial pressure of gaseous oxygen in the blood stream, which is coursing through a particular blood vessel in the body. The use of the probe is based on the principle of dye fluorescence oxygen quenching. Structurally the probe comprises two 150-micrometer strands of plastic optical fiber ending in a section of porous polymer tubing serving as a jacket or envelope for the fibers. The tubing is packed with a suitable fluorescent light-excitable dye placed on a porous adsorptive particulate polymeric support. The tubing or jacket is usually made of a hydrophobic, gas-permeable commercial material, known as Celgard, but other suitable hydrophobic gas-permeable material could be used for such structure. The fiber optic probe of the invention is of very small size and flexible so that it can easily be threaded through small blood vessels which are located in a variety of tissues of the body.

Fiber optic P.sub.O.sbsb.2 probe

A fiber optic pH probe suitable to be implanted in tissue for physiological studies is disclosed. The probe includes an ion permeable membrane envelope which encloses the ends of a pair of optical fibers. A pH sensitive dye indicator composition is present within the envelope. The probe operates on the concept of optically detecting the change in color of a pH sensitive dye.

Fiber optic pH probe

The preparation of dye-containing compositions, in which the dye is copolymerized with an acrylic based monomer, is disclosed. A hydrophilic copolymer is formed in which the dye is present in a nondiffusible form. The dye-acrylic polymer composition can be produced in the form of microbeads by emulsion polymerization. In addition, light scattering particles may be added in the form of a material such as polystyrene microspheres.

Dye-containing polymer composition

A method and apparatus for cutting cylinders of gelatinous material such as polyacrylamide gels into discs of precise thickness comprises a cutting surface which is placed in contact with the entire circumference of the cylinder. Cutting force is then applied from all points on the circumference at once, resulting in cutting the gel cylinder towards the center from all points on the circumference. This is accomplished by the use of a multi-bladed diaphragm similar to an iris diaphragm used for camera aperture control. The edges of the blades of the diaphragm form a variable dimension cutting surface.

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Falls Church, VA, United States of America

John I Peterson