Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
2002-12-31
2004-03-09
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S164000, C435S040500, C435S040520
Reexamination Certificate
active
06703242
ABSTRACT:
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
SEQUENCE LISTING, A TABLE, OR COMPUTER PROGRAM LISTING COMPACT DISK APPENDIX
Not Applicable
BACKGROUND OF THE INVENTION
The invention relates to a method of visualizing morphological structure in the nervous tissue. Despite enormous progress in the development of new techniques for studying central nerve pathways, few methods are available for visualization of the whole fiber architecture in the neural tissue. The most known methods for myelin sheath staining are iron-haematoxylin (Weil, 1928; Anderson, 1929), Luxol fast blue (Kluver and Barrera, 1953), Sudan-black (Olson and Traub, 1990), and a labeling with Black-gold (Schmued and Slikker, 1999). Another popular technique is axonal silver staining (Bodian; 1937, Davenport, 1929; Glees, 1946; Nauta, 1950; Beltramrino et al., 1993). In the last decades, methodological research primarily focused on tracing: single axonal pathways with markers moving by axonal transport (Kristensson and Olsson, 1971; Gerfen and Sawchenko, 1984; Katz et al, 1984; Schmued and Fallon, 1986; et al.) or with lipid soluble dyes drifting within the cell membrane (Honig and Hume, 1986).
While these and other techniques proved to be beneficial, they have certain limitations. Firstly, most known methods involve some kind of staining or dye incorporation. Secondly, they are rather sophisticated, labor intensive and time consuming. In addition, many of them require expensive reagents. Finally, the absolute majority of known tract tracing techniques deal with relatively thin (less than 60 &mgr;m) sections of neural tissue. A major drawback of thin sections is that they are virtually two-dimensional, and many sections should be pooled together for three-dimensional reconstruction of large segments of the fiber system in the CNS. Several studies dealing with 400-500 &mgr;m brain slices are focused on the morphology of single neurons pre-filled by intracellular injection of dye (Grace and Llinas, 1985; Hermes et al., 1996).
SUMMARY OF THE INVENTION
The purpose of this invention is to provide an extremely simple morphological technique for visualization of axonal pathways in thick sections of the nervous tissue.
To overcome the limitations of many previously developed methods, I chose an approach that utilizes thick (more than 200 &mgr;m) sections of the nervous tissue but does not involve staining. After years of experimentation, I have developed a technique, by which the whole fiber architecture of a thick slice of nervous tissue is made visible without using any dyes or other foreign substances.
This invention is based on a discovery that axonal bundles can be made visible without any staining, solely using their ability to deflect and deviate light rays directed at an angle exceeding the collecting angle of the objective lens (Senatorov 2002
a
and 2002
b
). The main principle of the invention is to make neural tissue transparent under normal (bright-filed) light, and then use the ability of axonal pathways to scatter light for observation using dark-field illumination.
One of the most attractive features of this invention is that even the three-dimensional structure of the whole white matter in the nervous tissue slice can become clearly visible (FIG.
1
A). Due to its extreme simplicity, the invention can be applied using basic microscopic equipment and a minimal set of reagents. As the present invention does not involve any kind of staining or dye incorporation, it does not require any expensive reagents and it consumes only a minimal amount of labor and time.
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patent: 6465208 (2002-10-01), Rogers
patent: 6472216 (2002-10-01), Chiang
Price PG, Fisher AW. Location of single neurons containing HRP for electron microscopy. Brain Res. Bull., 1977, 2: 495-497.
Grace AA, Llinas R. Morpholigical artifacts induced in intracellulary stained neurons by dehydration: Circumvention using rapid dimethyl sulfoxide clearing. Neuroscience, 1985; 16:461-475.
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Senatorov VV. Dark-field microscopy visualization of unstained axonal pathways using oil of wintergreen. J. Neurosci. Meth., 2002, 113, 59-62.
Senatorov Vladimir V.
Wallenhorst Maureen M.
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