Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
1999-12-06
2004-06-01
Chin, Christopher (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S006120, C435S007210, C435S007230, C435S040520, C436S177000, C382S133000, C428S346000, C428S352000
Reexamination Certificate
active
06743601
ABSTRACT:
This invention relates to a new method of laser capture microdissection. Generally, laser capture microdissection (here after also known as LCM) relates to a process of gathering samples of a specimen from a slide after visualization. In the process, an activatable surface is placed overlying the visualized specimen at a selected area, the surface activated to adhere to the specimen, and the activatable surface removed with the selected portion of the specimen attached.
In this disclosure of a new method of LCM, a physical process and designs for a thermally-activatable polymer layer are described in which the polymer on activation expands significantly in order to “reach out and grab” the target object within the microscopic field of interest. On cooling the activatable layer contracts (either during cooling or later in response to stored elastic stress) thereby bringing the “captured” microscopic target back closer to the original polymer surface. Using these physical processes and specific designs, “non-contact LCM” can be practiced by placing the selectively activatable coating at fixed separation distance (on the order of 5 to 20 microns) from the specimen, such as a tissue sample mounted on a slide. When a specimen has material selected from microdissection (typically by visualization within a microscope by transmission or epi-fluorescence) and the activatable coating activated with laser radiation, expansion and subsequent contraction of the activatable coating causes precise capture and extraction of the targeted elements within the sample. Activation may cause the surface to become adhesive (e.g., thermoplastic or photochemical bonding). In an alternative design, the specimen exposed side of the activatable coating can have a thin surface layer with the activatable coating forming a subsurface layer between a supporting substrate and the thin surface layer exposed to the specimen. The outward expansion of the subsurface layer brings the thin surface layer into contact with a complex tissue surface. In this alternative design, the surface layer can exhibit molecularly specific affinity for specific targets providing bonding specificity in addition to that of targeting the material selected for microdissection from the specimen.
BACKGROUND OF THE INVENTION
Microdissection of particular objects within a microscopic field have long been practiced in order to isolate specific elements from a complex field. This has been particularly important in complex biological samples where specific cells (e.g., stem cells) or clusters of cells (e.g., glomerulus from kidney) or even subcellular elements (such as a metaphase chromosome or a specific band of a chromosome) might be desired for subsequent biochemical analysis. Flow cytometry and cell sorting has been used for more than two decades to isolate specific populations of cells from single cell suspensions. In 1976, Meier-Ruge et al. described a pulsed UV-laser microscope, which was used to cut around the edges of invasive cancer cells in a tissue specimen in order to perform enzymatic activity analysis of such tissue. Schindler and Holland [patents] described a laser microscope used to isolate specific living cells from cell culture by either killing unwanted cells by a scanned laser beam or by cutting out all regions except those desired. Shibata et al. [Am. J Pathol. 141:539, 1992] described a similar process in which a simple UV-absorbing mask placed over specific microscopic regions of interest on a complex tissue section and the DNA in all other regions was damaged by UV irradiation. The small amounts on DNA in the protected regions could be extracted and amplified by polymerase chain reaction (PCR) in order to assess specific mutation in cancer cells within the tissue. Whetsell et al. [Oncogene 7:2355, 1992] described a variety of manual microdissection techniques applied to isolation and subsequent molecular analysis of pure populations of cells within the complex pathology specimen: including scraping target region with a needle or micropipette tip, injecting a fluid to make a cell suspension and then drawing the suspended microsample back into the tip for subsequent macromolecular analysis. In U.S. patent application Ser. No. 08/203,780 entitled Isolation of Cellular Material Under Microscopic Visualization by Lance A. Liotta, et al. filed Mar. 1, 1994, the idea of adhering visualized material on a specimen to a probe tip and then removing the tip with the procured sample to place in a solution for molecular analysis was described.
In U.S. patent application Ser. No. 08/544,388 entitled Isolation of Cellular Material Under Microscopic Visualization by Lance A. Liotta, et al. filed Oct. 10, 1995, the concept of microdissecting cancer cells in order to construct cDNA libraries of genes that are specifically expressed in those pure populations was disclosed. Two specific microdissection concepts were proposed: 1) manual “needle” microdissections and 2) focal activation by a light (laser) beam of an activatable bonding layer placed in contact with the tissue sample. The later concept has been developed into what is called laser capture microdissection [Emmert Buck et al. Science 274:998, 1996 and Bonner et al. Science 278:1481, 1997]. In that disclosure, microscopic visualization of a specimen occurred to select tissue for extraction. Thereafter, a film containing an activatable coating was placed on the specimen and activated by laser to a state where it adhered to the specimen at the selected material. When the activated film was removed, the adhered portion of the specimen was likewise removed effecting the desired dissection.
In this original LCM concept, the specificity of LCM is conferred by the focal bonding, which only occurs when a targeted region of the activatable film is activated. All other portions of the coating placed on the tissue sample were assumed to be nonbonding. In practice, tissue pathology sections presented irregular surfaces on a microscopic layer, and the thermoplastic polymer used for focal laser bonding to the targeted tissue sites can pick up peaks on the tissue surface or whole regions not strongly bonded to the underlying microscope slide. This problem led to the development of activatable polymer films that could be selectively cut or punched out in those regions where targeted transfers had occurred, thereby reducing dramatically non specific contamination arising from the large regions of the “transfer film” not activated by the laser.
In prior art with thermoplastic polymers used to create thermally activated bonds between two surface [here, the film substrate and the tissue section], it is known that the bond strength is dependent on applied pressure, fluidity of the melted polymer and time of activation. Thus the necessity for a strong bond to the tissue [i.e., stronger than the tissue bond strength to the glass microscope slide] would seem to require strong contact pressure or long activation pulses. In U.S. Provisional Patent Application Serial No. 60/094,871, filed Jul. 30, 1998 entitled PRECISION LASER CAPTURE MICRODISSECTION USING SHORT PULSE LENGTH by Robert F. Bonner, et al. it was disclosed that short pulses are required for and allow making the smallest LCM microdissections (e.g., <10 microns in diameter).
It is inherent in the Liotta 1995 disclosure that either the film is first contacted with the specimen and then activated [as described in Emmert-Buck et al. and in Bonner et al.] or alternatively, the activated region of the film is initially a short distance away from the specimen (microscopic) and only comes into contact with the specimen when activation occurs. However, in the original Liotta 1995 disclosure, there is always some part of the film in contact with the specimen. It is important to note that there is no suggestion of a deliberate spacing of all of the activatable coating from the specimen to obtain greater precision in the desired microdissection. In either event, the selected portion
Bonner Robert F.
Goldstein Seth R
Pohida Thomas
Smith Paul D.
Chin Christopher
Gabel Gailene R.
Hynes William Michael
The United States of America as represented by the Department of
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