Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
1998-10-02
2001-05-15
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
Reexamination Certificate
active
06232092
ABSTRACT:
BACKGROUND OF THE INVENTION
The study of tissues for medical research, disease diagnosis, disease prognosis as well as the examination of tissues after in vivo or in vitro exposure to agents of interest requires the ability to examine the intact tissue in three dimensions at all depths. For example, it is useful to be able to examine an entire lobe of an animal's lung where the animal has been exposed to an inhaleable substance, such as insulation fiber, in order to determine the distribution, biopersistence and pulmonary toxicity of the substance in the lung tissue.
Whole mount imaging of organisms or tissues provides an immediate and three-dimensional view of the architecture and morphology of the organism or tissue. Current technology in confocal microscopy and deconvolution technology allow the visualization of different focal planes within a sample of interest. Using these techniques, a three dimensional reconstruction of a biological sample can be made. However, these technologies are limited to relatively thin sections of sample. Under normal imaging conditions, the depth of optical penetration into specimens is typically limited to 20-40 microns.
Three-dimensional information can also be gained by serial section analysis, but only following an indirect, time-consuming process. In addition, sectioning is subject to many preparation artifacts that can distort the morphology of the tissues under study and disrupt or distort the distribution of the agent of interest within the tissue.
The methods used to achieve controlled exposure of animals to substance of interest often introduce method specific artifacts, necessitating the use of more than one method of exposure in any given study. Current fiber imaging methodologies, such as ash analysis, limit important observations and interpretations to bulk fiber load and size class of fibers, and do not reveal fiber position or associations with relevant pulmonary structures, important to lung pathology. Furthermore, traditional methods, such as ash analysis, can destroy pertinent information, while other methods, such as serial sectioning can take a year or more to complete thorough analysis of a specimen.
For example, pulmonary toxicity of natural and man made fibers are assessed in animal models using two-exposure methods: aerosol and intratracheal instillation. While each has distinct advantages, under certain conditions, one method may be more appropriate than the other. When the precise dose of a material is required, intratracheal instillation may be the preferred method of administration. However, the possibility of uneven distribution induced by fiber agglomeration may cloud interpretation of results. Although many studies have addressed clearance of fibers in lungs, none have adequately addressed the problem of fiber agglomeration in airways resulting from the method of fiber administration to lungs.
SUMMARY OF THE INVENTION
The present invention relates to the preparation of biological tissue for microscopic evaluation of morphologic features such as tissue architecture. The present invention allows the visualization of morphologic features of cells and tissues within intact biological specimens at an unlimited depth of observation. The present invention enables examination and analysis of man made and/or naturally occurring foreign material contained within cells, tissues or organ structures without physical destruction or cutting of the specimen itself.
The present invention eliminates light scattering properties of opaque tissue surrounding the optical plane of focus of the imaging plane. Under normal imaging conditions, depth of optical penetration into specimens is limited to 20-40 microns. The method of the present invention allows a depth of penetration of at least 800 microns. Therefore, in the example of rat lung tissue, entire airways within a lobe can be directly observed without physical disturbance. The present invention allows the examination of entire airways anywhere within intact lung lobes. The present invention relates to a method for clarifying an intact biological specimen, wherein the biological specimen is rendered suitable for analysis. The method comprises the steps of staining the specimen, and equilibrating the stained specimen in a clearing solution having a refractive index approximately that of the specimen.
The present invention relates to a method of clarifying lung tissue, wherein the lung tissue is rendered suitable for analysis. The method comprises the steps of staining the lung tissue with Lucifer Yellow CH and equilibrating the lung tissue in a clearing solution, whereby the refractive index of the clearing solution approximates that of the lung tissue. The present invention reveals the position of foreign material and or association of such material with relevant, morphology in a biological tissue of interest.
The present invention allows the determination of distribution quantity and size of exogenously derived material present in an intact biological specimen. The method comprises the steps of staining the specimen, equilibrating the specimen in a clearing solution, whereby the refractive index of the clearing solution approximates that of the specimen and visualizing the exogenously derived particles.
The present invention allows the determination of distribution, quantity and size of exogenously derived fibers present in an intact lung specimen wherein the specimen is rendered suitable for analysis. The method comprises the steps of staining the lung specimen with Lucifer Yellow CH, equilibrating the lung specimen in a clearing solution, whereby the refractive index of the clearing solution approximates that of the lung specimen and visualizing the exogenously derived fibers within the lung specimen.
Fibers as small as about 2 microns can be detected and measured using the method of the present invention.
The present invention is an improvement over other procedures such as fluorescently labeled antibodies and color dyes such as haematoxilyn and eosin. For example, antibodies are exquisitely specific to particular epitopes expressed in or on cells and tissues and would not necessarily reveal morphologic features. Color dyes interfere with light transmission and, therefore, prevent viewing of subsurface tissue structure. The present invention requires far less time than serial sectioning and unlike ash analysis or serial sectioning, does not result in the destruction of the tissue.
The present invention uses non-specific fluorescent dyes such as Lucifer Yellow CH or Nile Red that bind to common cellular constituents and provide a suitable contrasting agent while under illumination by specific wavelengths of light.
REFERENCES:
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patent: 6017725 (2000-01-01), Hoffmann et al.
Becker, David L. et al., “Enhancing The Laser Scanning Confocal Microscopic Visualization of Lucifer Yellow Filled Cells In Whole-Mounted Tissue”Scanning Microscopy,vol. 5, No. 3 pp. 619-624 (Sep. 1991).
Zill,Sasha N., et al., “Characterization of Insect Sense Organs And Optical Clearing Of Whole-Mount Preparations Using diI In Fixed Tissues”,J. exp. Biol.,175, pp. 299-303 (1993).
D.L. Gard, “Organization, Nucleation, and Acetylation of Microtubules inXenopus laevisOocytes: A Study by Confocal Immunofluorescence Microscopy,”Development Biology,143:346-362, (1991).
R.A. Rogers, et al., “3-D Confocal Microscopic Imaging of Amosite Fibers in Hamster Lungs,” Sixth International Meeting on the Toxicology of Natural and Man-Made Fibrous and Non-Fibrous Particles, Sep. 15-18, (1996) (Abstract).
R.A. Rogers, et al., “Enhanced Contrast within Plastic Embedded Tissue by Lucifer Yellow CH—An Ideal Stain for Laser Scanning Confocal Microscopy,”Molecular Biology of the Cell,3(Suppl):185a(Sep. 1992).
R. Rogers, et al., “3-D Imaging of Respirable Size Asbestos Fibers in Rat and Human Lung Tissue,” Proceedings Microscopy and Microanalysis 1995, p. 1018-1019, (Aug. 13-17, 1995).
M.W. Klymkowsky, “Whole-Mount Staining of Xenopus and Other Vertebrates,”Methods in Cell Biology,36:419-
Gitomer Ralph
Hamilton Brook Smith & Reynolds P.C.
Rogers Imaging Corporation
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