WO2005006051A1

WO2005006051A1 - Liquid cover slip for protection of tissue section on slide from macromolecule-degradation and for improvement of optical resolution, composit for producing of the same, structure of slide having the same and method for manufacturing the same

Info

Publication number: WO2005006051A1
Application number: PCT/KR2004/001742
Authority: WO
Inventors: Jung Young Lee; Nam Jin Yoo; Won Sang Park; Sug Hyung Lee
Original assignee: Jung Woo International Co., Ltd
Priority date: 2003-07-14
Filing date: 2004-07-14
Publication date: 2005-01-20
Also published as: KR100494979B1; KR20050008237A

Abstract

Disclosed are a liquid cover slip for protecting tissue slices and improving resolution, a composition for making the liquid cover slip, a slide structure having the cover slip and its manufacturing method. The slide structure includes a support layer, a tissue slice seated on an upper center of the support layer, and a liquid cover slip formed to surround an exposed surface of the tissue slice completely and made of a polyvinyl pyrrolidone polymer having a molecular weight of 10,000 to 360,000 as a main component. Thus, optical resolution is improved to enable micro observation of a tissue slice and precise selection of a micro region. In addition, the tissue slice keeps a non-aqueous state to be isolated from the air, so macromolecules in the tissue slice are not destructed or denatured.

Description

TITLE LIQUID COVER SLIP FOR PROTECTION OF TISSUE SECTION ON SLIDE FROM MACROMOLECULE-DEGRADATION AND FOR IMPROVEMENT OF OPTICAL RESOLUTION, COMPOSITE FOR PRODUCING OF THE SAME, STRUCTURE OF SLIDE HAVING THE SAME AND METHOD FOR MANUFACTURING THE SAME

TECHNICAL FIELD The present invention relates to a liquid cover slip for protecting tissue slices and improving resolution, a composition for making the liquid cover slip, a slide structure having the cover slip and its manufacturing method. More particularly, the present invention relates to a liquid cover slip for improving optical resolution of a tissue slice loaded on a slide substrate, which is observed by an optical microscope of a micro-ablating device and ablated by a laser beam, and also improving material preservation by preventing destruction of macromolecules in the tissue slice such as DNA, RNA and protein, a composition for making the cover slip, a slide structure having the cover slip and its manufacturing method.

BACKGROUND ART Generally, in order to observe a specific tissue slice of an organism tissue, a slide is made in advance through many steps. In more detail, the slide is made by a fixing step for collecting a part of necessary tissue from an organism source such as an animal, plant and microbe and then fixing a tissue slice, a dehydration step for removing moisture remained in the tissue slice, a clearing step for replacing alcohol penetrated into the tissue slice during the dehydration step with xylene, and an embedding step for including a paraffin solution into the tissue to make the tissue into a paraffin block. After that, a section step for sectioning the paraffin block into a predetermined thickness is conducted, and, after the tissue slice sectioned into a thin film shape is loaded on a slide, a staining step for dyeing intracellular components by using a predetermined dye is conducted for microscope observation. Meanwhile, in case of making a thin tissue slice with a crymicrotome, a tissue slice for observation using a microscope may be rapidly made without conducting the embedding step for making the tissue slice into a paraffin block form. The tissue slice adopted in embodiments of the present invention may be prepared not only in a paraffin block form but also in a frozen section form made using the crymicrotome. However, the conventional tissue slice making process a drawback that macromolecules in the tissue may be damaged during the process, particularly in the fixing step and the paraffin embedding step, though it gives good morphologic presentation. Meanwhile, in case of making a frozen section with the crymicrotome, the fixing step and the paraffin embedding step are not required, so macromolecules in the tissue may be well preserved but the morphologic preservation is bad. On the other hand, the frozen section making method has a drawback that an ablated tissue should be acquired newly, though it gives good preservation of macromolecules. In addition, the paraffin block made in the method allows easy acquiring of samples since each hospital has it sufficiently. Thus, one should be sufficiently consider advantages and disadvantages of each method in order to select a desired one among the aforementioned tissue slice making methods for a micro-ablating technique. In the micro-ablating technique which selectively ablating and collecting a specific cell or a specific region in the tissue slice observed by an optical microscope and then studying a genetic change or following researches for DNA, RNA or protein extracted from the cell, there are obviously required that (1) precise observation for the tissue slice using an optical microscope should be set forth in advance, (2) a specific region in the tissue slice should be precisely selected, (3) the selected region should be easily ablated, (4) the ablated tissue slice should be easily collected, and (5) destruction of macromolecules in the ablated tissue slice should be prevented during the micro-ablating procedure so that the following researches for DNA, RNA or protein extracted from the cell are not hindered. However, after the staining step is completed for the tissue slice loaded on the slide, mounting media showing a refractive index similar to the tissue slice itself are dropped on the upper surface of the slide, a cover glass is covered up in order to prevent light scattering, which is apt to arise in the tissue slice, thereby improving optical resolution, and then the tissue slice is observed using an observation tool such as an optical microscope, in the conventional art. However, in order to micro-ablate a specific region of the test piece loaded on the slide by using a laser beam in a visual field of the microscope in addition to the simple optical observation purpose, the micro ablation has been conducted by dyeing the slice tissue without treatment of a cover glass or a mounting medium and then observing the dried tissue slice since it is impossible to ablate a desired cell with a laser by means of the conventional method that the cover glass is loaded on the upper surface of the slide. Thus, since the micro ablation has been conducted without compensation or improvement of the optical resolution, lesion with a minute change such as precancerous lesion is not sufficiently distinguished or identified in the visual field of the microscope. Hereinafter, a principle of a conventional micro-ablating equipment is described in brief in order to specifically investigate factors, possibly causing problems while the micro ablation is progressed using the conventional micro-ablating equipment. The micro-ablating equipment using laser may be classified into two types. One is using a heat of laser, and the other is using a laser as a knife. The method using a heat of laser is currently realized in an A company in USA, which loads a dyed tissue slice on a glass plate slide, and then covers an upper portion of the tissue slice with a transparent thermoplastic film. Subsequently, if a laser beam is radiated above a cell region while the tissue slice is observed by an optical microscope, a lower surface of the thermoplastic film is melted a little to possess viscosity, and is then bound to the selected cell region of the tissue slice disposed below it. After that, if the thermoplastic film is lifted up, a desired cell is selectively collected on the lower surface of the thermoplastic film which is radiated by the laser beam. The method using a laser beam as a cutting tool, or a knife, is commonly employed in P, L and M companies in German, wherein P and L companies use a UV laser beam with a wavelength of 337 nm, while M company uses a UV laser beam with a wavelength of 350 nm. In all of the companies, the photolysis phenomenon of laser is used to precisely cut a thin film or tissue slice. The micro-ablating equipment of P company spreads a thin polyethylene naphthalate (PEN) film flatly and then adheres it along its edge by adhesive. After that, the tissue slice is loaded on an upper center of the thin PEN film, and then dyed. Subsequently, a region of a desired cell or cell group is selected as a closed curve through the microscope observation for the tissue slice, and then a laser beam whose focus is accurately controlled is radiated to the selected region along the closed curve. Photolysis happens in the material layer, that are the tissue slice the PEN film, radiated by the laser beam, so they are precisely ablated. If the first laser beam for ablation is completely radiated, a focus depth is adjusted differently from the focus in the laser ablation, and then a second laser beam for leaving the ablated region from a remained region is radiated. The second laser beam is radiated to the ablated region after the focus depth is adjusted so that the focus of the laser beam is not formed in the ablated region, differently from the focus depth of the first laser beam. Since the second laser beam is radiated with a deviated focus, photolysis does not happen any more in the ablated region, but a photonflow of the laser beam affects a certain catapulting power on the ablated region. Thus, if the ablated region ablated by the catapulting power of the laser beam is separated from the remained region and deviated upward, the ablated region is attached to a collection tube coated with mineral oil and positioned above it, thereby collecting the ablated region. However, this method has a drawback that the second laser beam may cause photolysis in the ablated region even a little since a distance between a focusing portion for photolysis and a defocusing portion adjusted for the catapulting effect is too short. L company proposes a micro-ablating equipment in order to solve the problem of the micro-ablating equipment of P company. The micro-ablating equipment of L company ablates a selected region of the tissue slice with a laser, then allows the ablated region to free falls down, and then disposes the collection tube below the slide in order to solve the partial photolysis problem which happens when the laser beam is secondarly radiated to upwardly separate the tissue slice sliced by the catapulting effect. However, the collecting method using the free falling of the ablated region has a problem that its collecting rate is deteriorated due to minute static electricity generated in the equipment. The equipment of M company adopts a principle similar to that of L company, but it stamps the micro-ablated region with an adhesive cap from above in order to collect the ablated region, instead of using the free falling. However, this method requires many addition processes for the collecting work, and it is very cumbersome to take up and use the collected tissue slice. Reference may be made in relation to the micro-ablating equipment and the observation, ablation and collection methods using the equipment, particularly

US Filing No. 09/043,093, WO 99/39176, WO 00/34757, US 6,010,888 and so on. However, all of them cannot give improvement of optical resolution due to the limit that a cover class may not be covered during the micro-ablating process, so it is apparently understood in the art that there is a technical limitation in the problem that minute observation and selection of an ablated region are not easy.

DISCLOSURE OF THE INVENTION On the ground of the problems of the prior art, the invention is directed to accomplishing the following technical subjects by one effort that (1) optical resolution of a tissue slice is sufficiently improved during the observation using a microscope, (2) the tissue slice is easily ablated by an ablating laser beam, (3) the tissue slice is easily separated after ablation so as not to give any effect on the process of extracting RNA, DNA and protein from the ablated tissue slice, (4) denaturation of macromolecules in the tissue slice is restrained, and (5) it is used and handled in an easy way. In order to obtain these technical subjects, an object of the present invention to provide a liquid cover slip for protecting a tissue slice and improving resolution, a composition using the cover slip, a slide structure made using the cover slip and its manufacturing method. In order to achieve the object, the present invention provides a liquid cover slip for protecting a slide tissue slice and improving resolution, which is made of polyvinyl pyrrolidone having a molecular weight of 10,000 to 360,000 as a main component. At this time, a small amount of Congo red or enhancing UV absorption effeect is preferably added and uniformly dispersed into a coating layer made of the polyvinyl pyrrolidone as a main component. In another aspect of the invention, there is also provided a composition for making a liquid cover slip for protecting a slide tissue slice and improving resolution, which includes an alcoholic solvent; a polyvinyl pyrrolidone polymer having a molecular weight of 10,000 to 360,000; and Congo red as a UV absorption enhancer, wherein the polyvinyl pyrrolidone polymer is included in the alcoholic solvent as a main component, and a small amount of Congo red is dissolved therein. Preferably, the alcoholic solvent is ethanol or isopropanol. Particularly, it is more preferable that the polyvinyl pyrrolidone polymer is 5 to 15 wt% on the basis of a weight of the alcoholic solvent, and the Congo red is 0.0025 to 0.024 wt% on the basis of the weight of the alcoholic solvent. In still another aspect of the invention, there is also provided a slide structure having a liquid cover slip for protecting a slide tissue slice and improving resolution, which includes a support layer; a tissue slice seated on an upper center of the support layer; and a liquid cover slip formed to surround an exposed surface of the tissue slice completely and made of a polyvinyl pyrrolidone polymer having a molecular weight of 10,000 to 360,000 as a main component. At this time, the support layer is preferably a polymer film layer with a plate structure. Meanwhile, it is more preferable that a slide substrate interfaced with a lower surface of the polymer film layer and attached thereto by an interlayer adhesive member is further provided. In addition, the slide substrate preferably has a predetermined open window, which provided right below a plane region including a region occupied by the tissue slice seated on the polymer film layer. Also preferably, the polymer film layer is made of polyethylene naphthalate polymer as a main component. In further another aspect of the invention, there is also provided a method for manufacturing a slide structure having a liquid cover slip for protecting a slide tissue slice and improving resolution, which includes (a) attaching a polymer film layer with a plate structure to an edge of an upper surface of a slide substrate having a window by means of a viscous member; (b) seating a prepared tissue slice on a center of an upper surface of a polymer film layer attached to the upper surface of the slide substrate to which a window lead is combined; (c) removing the window lead from the slide substrate and then dyeing the tissue slice seated on the slide substrate; and (d) forming a liquid cover slip to cover an exposed surface of the tissue slice completely. At this time, the slide substrate of the step (a) is preferably made of a material in which a carbonate material is included as a main component, and polyetherimide and a static electricity inhibiter are slightly added. Meanwhile, the polymer film layer of the step (a) is preferably made of a polyethylene naphthalate polymer as a main component. The liquid cover slip of the step (d) is preferably made of an alcoholic solvent such as ethanol and isopropanol; a polyvinyl pyrrolidone having a molecular weight of 10,000 to 360,000; and Congo red. At this time, more preferably, the polyvinyl pyrrolidone polymer is 5 to 15 wt% on the basis of a weight of the alcoholic solvent, and the Congo red is 0.0025 to 0.025 wt% on the basis of the weight of the alcoholic solvent. To claim 11 , wherein the tissue slice used in the step (b) is a frozen section. Hereinafter, embodiments are proposed for specific explanation of the present invention with reference to the accompanying drawings when required for better understanding of the invention. However, the embodiments of the present invention may be modified in various ways, and the scope of the invention should not be interpreted as limited to the embodiments. The embodiments of the present invention are just given in a way of giving better illustration to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features, aspects, and advantages of preferred embodiments of the present invention will be more fully described in the following detailed description, taken accompanying drawings. In the drawings: FIGs. 1 to 3 are photographs for comparing embodiments of the present invention with comparative examples in order to explain the difference of optical resolution; FIGs. 4 and 5 are photographs showing results of the chromatography experiment for the comparative examples and the embodiments of the present invention, in which a destruction degree of RNA included in a tissue slice is observed along with time; FIG. 6 shows an absorption range for each wavelength of Congo red added into a liquid cover slip according to the present invention; FIGs. 7 and 8 are sectional views showing a slide structure having the liquid cover slip according to the embodiments of the present invention; FIG. 9 is a sectional view for illustrating micro ablation and collection related to the slide structure shown in FIG. 8; and FIG. 10 is a flowchart for illustrating a method for manufacturing a slide structure having a liquid cover slip according to the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION A polyvinyl pyrrolidone polymer used in the present invention is a polymer material having various molecular weights, which is known to be not harmful for the human body and soluble in both water and alcohol. Preferably, the polyvinyl pyrrolidone polymer is dissolved in a non-aqueous alcohol solvent and then used in order to form a liquid cover slip for wrapping and protecting a tissue slice and improving resolution. Since inner components of the tissue slice are generally apt to be easily damaged by atmosphere or moisture, the alcohol solvent is preferably used, and ethanol or isopropanol is more preferable. However, a coating made of polyvinyl pyrrolidone as a main component for protecting a slide tissue slice, which is used instead of a conventional mounting material or cover glass in the slide structure manufacturing procedure, not only plays a role of a protective layer but also prevents diffused reflection or scattering of light when being observed with an optical microscope since an irregular morphology of the tissue slice may be improved when an exposed portion of the tissue slice is surrounded by the coating layer. In addition, since a refractive index (1.43) of the polyvinyl pyrrolidone has no great difference from that of the tissue slice, the coating layer also ensures high resolution when being observed with an optical microscope. A coating composition used for making a liquid cover slip for protecting a tissue slice and improving resolution according to the present invention includes, with ethanol as a solvent, 10 wt% of polyvinyl pyrrolidone having a molecular weight of 40,000 on the basis of a criterion weight of the ethanol solvent, and 0.002 wt% of Congo red as an UV laser beam absorption enhancer on the basis of a criterion weight of the ethanol solvent. A slide structure is manufactured using the coating composition, and experimented for check improvement of optical resolution and preservation of the inner components of the tissue slice and the experiment results are examined. The improvement of optical resolution of the present invention is satisfactorily confirmed through specific experimental data, as described below in detail with reference to FIGs. 1 to 3. FIGs. 1 to 3 are photographs for comparing comparative examples and embodiments of the present invention in order to explain difference of optical resolution. Tissue slices are collected for observing colon cancer, hepatoma and gastric cancer of the patient respectively, and then prepared as frozen sections whose images are shown in FIGs 1 to 3 with being enlarged by an optical microscope. Referring to FIGs. 1 to 3, a right photograph on the basis of a central arrow in each figure shows a tissue slice observed with an optical microscope with a protective layer being coated thereon according to each embodiment of the present invention, while a left one of the central arrow shows a tissue slice according to each comparative example for comparison with the embodiment of the present invention. First, a predetermined dying step is conducted for tissue slices loaded on a slide, and then the tissue slices that keep exposed to atmosphere are observed with an optical microscope to obtain the photographs of the comparative examples. After that, the tissue slices are coated with a liquid cover slip for protecting the tissue slice and improving resolution according to the present invention, and then each tissue slice is observed with the optical microscope again to obtain the photographs of the embodiments of the present invention. Comparing the photographs in the right and left positions of FIGs. 1 to 3 according to the comparative examples and the embodiments, improvement of the optical resolution may be confirmed by intuition. That is to say, it is found that cells are not easily distinguished in the photographs of the comparative examples (1 A of FIG. 1 , 2A of FIG. 2 and 3A of FIG. 3) since the surface of the dyed tissue slice is not kept flat to cause scattering of light and thus the observed cells are shown in black. However, in the photographs of the embodiments, it is found that cancer cells and inflammatory cells are clearly distinguished (see 1 B of FIG. 1), it is also obviously seen that cancer cells form a linear structure (see 2B of FIG. 2), and it is also distinctly distinguished that cancer cells are diffusively dispersed (see 3B of FIG. 3). Thus, it is understood that the liquid cover slip of the present invention acts as a functional layer for improving optical resolution of the tissue slice on the slide. Meanwhile, another important role of the liquid cover slip according to the present invention is that it has presentation of macromolecules in the tissue slice. In particular, the frozen section doubles the effects. That is to say, since macromolecules in the tissue embedded in the paraffin after being fixed to a fixing solution are already destructed or partially denatured, the presentation of macromolecules may be treated somewhat meaningless. However, in the frozen section, macromolecules are well presented and various destructive enzymes such as Nrase, Dnase and Protease capable of destructing the macromolecules are also preserved, so the macromolecules may be destructed by the destructive enzymes if the frozen section is not treated carefully. Meanwhile, since the destructive enzymes are not active without substrate, that is water, the aforementioned problem may be solved if the multi-stage staining step is progressed for the frozen section in a non-aqueous state. However, if the stained frozen section keeps exposed to atmosphere while it is observed with an optical microscope and ablated by a laser beam, there is no way to prevent a little moisture included in the atmosphere from destructing the macromolecules as mentioned above. Thus, if the tissue slide is surrounded by the liquid cover slip for protecting tissue slices and improving resolution, made of a polyvinyl pyrrolidone as a main component, according to the present invention, the frozen section is isolated from the atmosphere, so the non-aqueous state of the frozen section may be kept on and the destruction of macromolecules in the tissue slice may be prevented. That is to say, in case the tissue slice, particularly a frozen section, is surrounded by the liquid cover slip for protecting tissue slices and improving resolution, made of a polyvinyl pyrrolidone as a main component, the frozen section is isolate from the atmosphere with keeping inner and outer regions of the frozen section in a non-aqueous state, so it may be understood that the liquid cover acts as a functional layer for improving preservation of the macromolecules in the tissue slice. In particular, the preservation improvement of the macromolecules in the tissue slice according to the present invention may be easily and satisfactorily evaluated through a specific experiment for RNA, which is known to be most easily destructed, as described in detail below with reference to FIGs. 4 and 5. FIGs. 4 and 5 are photographs showing chromatography experimental results according to comparative examples and embodiments in which destruction degrees of RNA included in a tissue slice are observed along time. In the figures, FIG. 4 is a photograph showing an experimental observation result of a tissue slice on which the liquid cover slip for protecting tissue slices and improving resolution is not formed, while FIG. 5 is a photograph showing an experimental observation result of a tissue slice protected by the liquid cover slip for protecting tissue slices and improving resolution according to the present invention. As seen in FIG. 4, among two pairs of bands including 28S band 4A and 18S 4B of ribosomal RNA extracted at each time from a frozen section which is positioned on a slide, stained, and then left alone at a room temperature without forming a separate outer coating layer, it is found that the 28S band 4A is particularly weakly expressed as time goes. That is to say, it may be checked that the 28S band 4A of RNA is remarkably weakened as time goes, compared with the initially observed band (0 time). This is included in the frozen section exposed outside. It is because RNA is easily destructed by moisture or the like contained in the atmosphere. To the contrary, as seen in FIG. 5, in case a tissue slice is coated with the liquid cover slip according to the present invention, it is found that both 28S band 5A and 18S band 5B of RNA are substantially kept even after 24 hours as they were at the initial stage (0 time). Thus, it may be understood that the preservation of macromolecules in the tissue slice is improved in the present invention. If the preservation of macromolecules in the tissue slice is improved, it apparently gives more preferable effects to the research for observation and micro ablation of a tissue slice on a slide for a middle or long period with the passage of time. Meanwhile, the polyvinyl pyrrolidone polymer has a weak energy absorbing ability of UV laser beam, so a high-energy UV laser beam is required for progress the ablating work with a UV laser beam. Thus, an additive for improving the energy absorbing ability of UV laser beam may be added in order to compensate it, which improves efficiency of the ablating work. For this purpose, a small amount of Congo red is preferably dispersed uniformly together with the polyvinyl pyrrolidone polymer for achieving preferable effects. Thus, a liquid composition for making a liquid cover slip for protection of slide tissue slice and improvement of resolution is prepared by dissolving a slight amount of Congo red used as a UV absorption enhancer together with a polyvinyl pyrrolidone polymer as a main component in an alcohol solvent, and then a coating layer is formed on the upper layer of the slide on which the tissue slice is loaded by means of various well-known ways. For example, the coating liquid (or, the liquid composition) is dropped on a side of the slide and the slide is inclined so that the coating liquid runs down thereon in order to form the coating layer; the coating liquid is dropped on a right upper surface of the tissue slice and then a constant rotating force is applied thereto in order to form the coating layer by means of centrifugal force; or the coating liquid is sprayed on the upper surface of the slide in order to form the coating layer. Meanwhile, the coating layer may be manually formed, but preferably formed using an automation system for stable and simple mass production. If the liquid cover slip for protection of tissue slice and improvement of resolution is coated on the upper surface of the slide and then left alone at a room temperature, the volatile alcohol solvent is mostly removed, and the polyvinyl pyrrolidone polymer in which Congo red is uniformly dispersed is changed into a solidified coating layer. This solidified coating layer may function as a protective layer for preventing destruction of macromolecules in the tissue slice since the tissue slice on the slide may be preserved for a long time. In addition, since the liquid cover slip contains a UV absorption enhancer such as Congo red having a relatively excellent energy absorbing ability of UV laser beam with a wavelength of 355 nm, the liquid cover slip according to the present invention may be more easily ablated with a small energy rather than one without Congo red. FIG. 6 is a graph showing an absorption range for each wavelength of Congo red added into the liquid cover slip according to the present invention. As shown in FIG. 6, X axis of the graph designates a wavelength range and Y axis designates absorbance, so a relative absorbance at a certain wavelength may be found in the graph. It is seen that two peaks are formed in the graph of FIG. 6, and a peak corresponding to the second intensity is formed at a wavelength range around 350 nm which is commonly used in a macro-ablating equipment. Thus, in case Congo red is added to the liquid cover slip according to the present invention, the absorbance of micro-ablating UV laser beam is enhanced, thereby making it possible to conduct the ablating work smoothly with a low energy state. The polyvinyl pyrrolidone polymer used for making the liquid cover slip for protection of slide tissue slice and improvement of resolution according to the present invention may have various molecular weights, and its effects purposed by the present invention may be obtained to the maximum when the molecular weight is in the range of 10,000 to 360,000 as mentioned above. If the molecular weight is not in the range, that is, if the molecular weight is less than 10,000, the liquid cover slip is cracked during the drying step after the alcohol component, a solvent of the polyvinyl pyrrolidone, is volatilized, and if the molecular weight exceeds 360,000, the liquid cover slip may not be easily ablated by UV laser beam, so it is not suitable particularly as a liquid cover slip for protection of slide tissue slice and improvement of resolution, employed in a micro-ablating equipment using UV laser beam. The coating composition used for making the liquid cover slip for protection of slide tissue slice and improvement of resolution according to the present invention includes a polyvinyl pyrrolidone polymer and Congo red, preferably 5 to 15 wt% of polyvinyl pyrrolidone polymer on the basis of the criterion weight of the alcohol solution and 0.0025 to 0.025 wt% of Congo red on the basis of the criterion weight of the alcohol solvent. If the content of the polyvinyl pyrrolidone polymer is less than 5 wt%, viscosity is decreased and thus the liquid composition is too easily run down when being dropped on the tissue slice, so the cover slip becomes too thin or occasionally some area may be left without being coated with the cover slip, thereby not sufficiently improving the surface form of the tissue slice. If the content exceeds 15wt%, the cover slip is formed too thicker than required due to high viscosity, thereby needing a high energy laser beam for ablation of the cover slip. Thus, in order to achieve the purpose of the present invention, the content of polyvinyl pyrrolidone should be controlled within the range. In addition, if the content of Congo red is less than 0.0025 wt%, UV absorption effect is not realized and thus a high-energy laser beam is required. On the while, if the content exceeds 0.025 wt%, a red color of Congo red itself is excessively manifested, which may give an influence on morphologic observation of the tissue slice. Thus, in order to sufficiently obtain the purpose of the present invention, the content of Congo red should be controlled within the above range. Now, a slide structure having the liquid cover slip for protection of slide tissue slice and improvement of resolution according to the present invention is described in detail with reference to FIGs. 7 and 8. FIGs. 7 and 8 are sectional views showing sectional views showing a slide structure having the liquid cover slip according to the embodiments of the present invention. FIG. 7 is a sectional view showing a slide structure having the liquid cover slip according to an embodiment of the present invention. Referring to FIG. 7, the slide structure includes a slide substrate 70 having a flat plate structure and acting as a support layer, and a polymer film 72 attached on the slide substrate 70 by means of an interlayer adhesive member (not shown), and a tissue slice 74 and a liquid cover slip 76 are laminated thereon. The slide structure shown in FIG. 7 allows detailed and precise observation of micro structure in the tissue slice due to the improved optical resolution of the liquid cover slip 76 according to the present invention in case it is used simply for observation using an optical fiber microscope. In addition, due to the improvement of reservation for macromolecules in the tissue slice, more reliability is ensured for the succeeding researches. Meanwhile, even in the case that a method for loading the slide structure shown in FIG. 7 on an ablating stage of the micro-ablating device without overturning it, radiating a laser beam from a lower position, and then collecting an ablated tissue slice upward is used, it is possible to ensure efficient and reliable selection of an ablation region due to the improved resolution for optical observation and gives better efficiency and reliability to the succeeding researches for DNA, RNA and protein extracted from the tissue slice due to the improved preservation of macromolecules in the ablated tissue slice. FIG. 8 is a sectional view showing a slide structure having the liquid cover slip for protection of slide tissue slice and improvement of resolution according to another embodiment of the present invention. Referring to FIG. 8, a polymer film layer 82 is attached to an interlayer adhesive member (not shown) on an upper edge of a slide substrate 80 with a window. A tissue slice 84 is seated on an upper center of the polymer film layer 82. A liquid cover slip 86 for surrounding the tissue slice completely is laminated thereon, thereby configuring the slide structure. The liquid cover slip 86 is closely adhered to the tissue slice 84 with completely surrounding its outside. The liquid cover slip 86 has a refractive index similar to the tissue slice and its interface contacted with air is formed flat so as to prevent diffused reflection or scattering of light when the tissue slice is observed with a microscope. In addition, since the tissue slice has an improved surface shape, optical resolution is sufficient even to a micro region. In addition, the liquid cover slip 86 prevents macromolecules in the tissue slice 84 such as DNA, RNA and various proteins from being damaged or lost with the passage of time, thereby ensuring reliability for the succeeding researches for the ablated tissue slice or DNA, RNA and protein extracted from the tissue slice. Meanwhile, the slide substrate 80 has a window so that the lower surface of the polymer film layer 82 on which the tissue slice 84 is seated is exposed directly to atmosphere. When the tissue slice 84, particularly a frozen section, is loaded on the polymer film layer 82, the low-temperature allogenic ization is generated due to convection and conduction while the polymer film layer 82 is contacted with the frozen section with an extremely low temperature, thereby deteriorating an adhesive property of the frozen section to the polymer film layer 82. Thus, in order to prevent this problem, the window of the slide substrate 80 is combined with a window lead 88 when the frozen section is loaded. A direction of attaching or detaching the window lead 88 to/from the slide substrate 80 is designated by an arrow 8A. On the other hand, it should be fully understood that the thickness of the slide structure shown in FIGs. 7 and 8 shows a wide difference from an actual one, and the thickness of each stage is somewhat exaggerated for easier illustration in a vertical structure, not based on an accurate reduced scale. Generally, the polymer film layer 72 and 82 has a thickness of 1 to 2 micrometers (μm), the tissue slice 74 and 84 has a thickness of 2 to 6 micrometers (μm), and the liquid cover slip 76 and 86 has a thickness of 8 to 22 micrometers (μm). In particular, it is apparently understood to those skilled in the art without any special explanation that polymer film layer, the tissue slice and the liquid cover slip have very small thickness, not easily expressed in the figure in comparison to the thickness of the slide substrate 70 and 80. FIG. 9 is a sectional view for illustrating the micro-ablating and collecting processes for the slide structure illustrated and described in FIG. 8. Referring to FIG. 9, the slide structure of FIG. 8 is turned over and loaded on a slide stage 90 of the micro-ablating device. Then, the tissue slice 84 is observed in a direction of the arrow 9A with an observation means, and an ablation region (not shown) is selected. After that, a laser beam is radiated in a direction of an arrow 9B from a right upper position of the slide structure which is loaded with being turned over so as to ablate the polymer film layer 82, the tissue slice 84 and the liquid cover slip 86 in the selected region. Then, due to the gravity applied to the completely ablated micro tissue region, the ablated region is collected in a direction of an arrow 9C into a collecting container 92 arranged right below it. Meanwhile, static electricity may be generated to the window slide substrate 80 due to the reasons that the micro-ablating device is operated by electricity, each layer of the slide structure is very thin, and the radiated laser beam has certain energy in itself. Accordingly, if there is generated static electricity greater than the gravity exerted to the ablated micro region, the ablated region is apt to be not adequately dropped into the collecting container 92. Thus, it is not desirable to depend only on the collecting method using gravity, but a static electricity inhibitor is also added to the material of the window slide substrate so as to eliminate minute static electricity generated in the slide substrate. At the same time, when the ablating work using a laser beam to the tissue slice selected as an ablation region is being completed, tension of the radiated laser beam is used to promote separation of the ablated region from the remained region. Accordingly, the collecting rate is not deteriorated due to the static electricity. Meanwhile, the slide structure may be loaded on the slide stage of the micro-ablating device as it is, not in a turned-over state as shown in FIG. 9. Though the slide structure is loaded as mentioned above, the processes of observing the tissue slice, ablating it with a laser beam and collecting the ablated tissue slice may be progressed in the aforementioned order. Among the components of the slide structure described with reference to

FIGs. 7 to 9, the liquid cover slip 76 and 86 for protection of tissue slice and improvement of resolution is preferably made of any of the above-described materials, and the polymer film layer 72 and 82 is preferably made of polyethylene naphthalate polymer. Meanwhile, the slide substrate 70 and 80 is preferably made of a material including carbonate as a main and polyetherimide and static electricity inhibitor as additives. In particular, in case of the slide substrate 80 having a window which is capable of using the drop-collecting manner in the micro-ablating procedure, adding the static electricity inhibitor may greatly improve the collecting rate of the ablated regions. A method for manufacturing a slide structure which is proposed to accomplish the object of the present invention is not described in detail with reference to FIG. 10. Here, the same reference numeral is given to the same component that may be referred to FIG. 9. FIG. 10 is a flowchart for illustrating the method for manufacturing a slide structure having a liquid cover slip for protecting a slide tissue slice and improving resolution according to the present invention. First, the polymer film layer 82 is attached on an upper edge of the slide substrate 80 with a window by means of a viscous member (not shown) (S100). At this time, the window lead 88 combined to the window of the slide substrate 80 may be attached to the polymer film layer 82 with being combined to the slide substrate 80 in advance. In addition, the window lead 88 may be combined after the polymer film layer 82 is attached on the upper surface of the slide substrate 80 in advance. Meanwhile, the slide substrate 80 is preferably made of material including carbonate as a main component and polyetherimide and static electricity inhibitor as additives, and the window lead is preferably made of high-functional engineering plastic, polyetherimide or polyarylate (commonly called 'ardel'). In particular, it is more preferable that the static electricity is added to the slide substrate 80 since the collecting rate of the micro-ablated tissue slices is not deteriorated by static electricity as mentioned in FIG. 9. Subsequently, the prepared tissue slice 84 is seated on the upper center of the polymer film layer 82 attached to the upper surface of the slide substrate 80 to which the window lead 88 is combined (S102). At this time, the polymer film layer 82 is preferably made of a material having polyethylene naphthalate as a main component. Meanwhile, since the polymer film layer 82 has a very small thermal capacity, low temperature coordination is progressed due to rapid heat transfer when the tissue slice 84 is contacted with the polymer film layer 84 in case the tissue slice 84 is a low temperature frozen section. Thus, the frozen section 84 and the polymer film layer 82 become in thermal equilibrium, thereby making it not smooth to contact both materials. Such problem is solved by the window lead 88. The window lead 88 interposes a thin air layer (not shown) with being closely adhered to the polymer film layer 82 so as to act as a buffer against the thermal equilibrium and block the surface exposed to the polymer film layer 82, thereby restraining rapid low temperature coordination of the polymer film layer 82. Thus, the temperatures of the polymer film layer 82 and the frozen section 84 may be kept to have a certain difference, so the frozen section 84 may be easily adhered and fixed to the polymer film layer 82. As a succeeding step, the window lead 99 is removed from the slide substrate 80, and the tissue slice 84 on the slide substrate 80 is stained (S104). This staining step may be progressed in various ways using various dyes well known in the art, which is apparent to those skilled in the art and not described here in detail. Finally, the composition for making the liquid cover slip for protection of slide tissue slice and improvement of resolution, made of polyvinyl pyrrolidone with a molecular weight of 10,000 to 360,000, preferably 40,000, as a main component, is coated to surround all of the exposed surface of the tissue slice, thereby forming a coating layer (S106). As a result, the procedure for manufacturing the slide structure according to one embodiment of the present invention is completed. At this time, the composition or making the liquid cover slip 86 for protection of slide tissue slice and improvement of resolution preferably includes an alcoholic solvent such as ethanol and isopropanol, and 5 to 15 wt% (preferably, 10 wt%) of a polyvinyl pyrrolidone polymer with a molecular weight of 40,000 on the basis of the weight of the alcoholic solvent, and 0.0025 to 0.025 wt% (preferably 0.02 w%) of Congo red is also added as a UV laser beam absorption enhancer on the basis of the weight of the alcoholic solvent. Meanwhile, it is apparent that the slide structure manufactured as mentioned above may continuously execute the observation/ablation step (S108) and the ablation/collection step (S110) using an optical instrument or a micro-ablating device on which an optical instrument is loaded, and the step of collecting the ablated tissue slice and then using it as a sample for succeeding researches (S112). At this time, the slice structure as shown in FIG. 7 may be loaded on the slide stage (not shown) with its upper and lower sides kept as originally or turned over for the observation and ablation work. In addition, in the ablation/collection step (S110), the tissue slice may be collected in the same direction as the radiated direction of laser beam or in an opposite direction in consideration of the used slide structure and the radiating direction of the laser beam that is an ablating means. Moreover, it is also apparent that the tissue slice may be collected using gravity or using a separate collecting device by giving an external force in a direction opposite to the gravity. The embodiments of the present invention have been described in detail. Here, the terms used herein are purposed for specific explanation of the invention, not for limiting the meaning or scope of the invention stated in the appended claims.

INDUSTRIAL APPLICABILITY The liquid cover slip, the composition for making the liquid cover slip, the slide structure having the cover slip and its manufacturing method according to the present invention give advantages as follows. First, micro ablation for a tissue slice and precise selection for the tissue slice are enabled due to improvement of optical resolution. Second, the problem that macromolecules in the tissue slice are exposed to ambient air and damaged by moisture is fundamentally solved, particularly when a frozen section is used. Thus, various macromolecules in the tissue slice are preserved well, so it is possible to ensure reliability for the following researches. In particular, since the composition for making the liquid cover slip is made in a non-aqueous state, it is possible to prevent endogeneous RNase that may exist in the tissue slice. Third, when works related to the micro-ablating device are progressed, it ensures simple collecting procedure and increased collecting rate. Fourth, though the composition for making the liquid cover slip is liquid, it is rapidly solidified to make a thin film shape together with evaporation of the alcoholic solvent when being coated on the tissue slice. In addition, since the used solvent is a volatile alcoholic solvent, it is not harmful to the human body, and internal components of the tissue slice (e.g., the macromolecules) may be easily extracted since it is soluble, thereby ensuring easily usage and treatment. Finally, when using the present invention, the enlarged image precisely observed by the improved resolution and the improved reservation of the macromolecules in the tissue slice may be transmitted to the third party, and sufficient and reliable opinions may be received from a person in a remote place.

These make it possible to prepare a fundamental technical base.

Claims

WHAT IS CLAIMED IS:

1. A liquid cover slip for protecting a slide tissue slice and improving resolution, which is made of polyvinyl pyrrolidone having a molecular weight of 10,000 to 360,000 as a main component.

2. The liquid cover slip according to claim 1 , wherein Congo red is added and uniformly dispersed into a coating layer made of the polyvinyl pyrrolidone as a main component.

3. A composition for making a liquid cover slip for protecting a slide tissue slice and improving resolution, comprising: an alcoholic solvent; a polyvinyl pyrrolidone polymer having a molecular weight of 10,000 to 360,000; and Congo red.

4. The composition for making a liquid cover slip according to claim 3, wherein the alcoholic solvent is ethanol or isopropanol.

5. The composition for making a liquid cover slip according to 3 or 4, wherein the polyvinyl pyrrolidone polymer is 5 to 15 wt% on the basis of a weight of the alcoholic solvent, and the Congo red is 0.0025 to 0.025 wt% on the basis of the weight of the alcoholic solvent.

6. A slide structure having a liquid cover slip for protecting a slide tissue slice and improving resolution, comprising: a support layer; a tissue slice seated on an upper center of the support layer; and a liquid cover slip formed to surround an exposed surface of the tissue slice completely and made of a polyvinyl pyrrolidone polymer having a molecular weight of 10,000 to 360,000 as a main component.

7. The slide structure having a liquid cover slip according to claim 6, wherein the support layer is a polymer film layer with a plate structure.

8. The slide structure having a liquid cover slip according to claim 7, further comprising a slide substrate interfaced with a lower surface of the polymer film layer and attached thereto by an interlayer adhesive member.

9. The slide structure having a liquid cover slip according to claim 8, wherein the slide substrate has a predetermined open window, which provided right below a plane region including a region occupied by the tissue slice seated on the polymer film layer.

10. The slide structure having a liquid cover slip according to any of claims 7 to 9, wherein the polymer film layer is made of polyethylene naphthalate polymer as a main component.

11. A method for manufacturing a slide structure having a liquid cover slip for protecting a slide tissue slice and improving resolution, comprising: (a) attaching a polymer film layer with a plate structure to an edge of an upper surface of a slide substrate having a window by means of a viscous member; (b) seating a prepared tissue slice on a center of an upper surface of a polymer film layer attached to the upper surface of the slide substrate to which a window lead is combined; (c) removing the window lead from the slide substrate and then dyeing the tissue slice seated on the slide substrate; and (d) forming a liquid cover slip to cover an exposed surface of the tissue slice completely.

12. The method for manufacturing a slide structure having a liquid cover slip according to claim 11 , wherein the slide substrate of the step (a) is made of a material in which a carbonate material is included as a main component, and polyetherimide and a static electricity inhibiter are slightly added.

13. The method for manufacturing a slide structure having a liquid cover slip according to claim 11 , wherein the polymer film layer of the step (a) is made of a polyethylene naphthalate polymer as a main component.

14. The method for manufacturing a slide structure having a liquid cover slip according to claim 11 , wherein the liquid cover slip of the step (d) is made of an alcoholic solvent; a polyvinyl pyrrolidone having a molecular weight of 10,000 to 360,000; and Congo red.

15. The method for manufacturing a slide structure having a liquid cover slip according to claim 14, wherein the alcoholic solvent uses ethanol or isopropanol.

16. The method for manufacturing a slide structure having a liquid cover slip according to claim 14 or 15, wherein the polyvinyl pyrrolidone polymer is 5 to 15 wt% on the basis of a weight of the alcoholic solvent, and the Congo red is 0.0025 to 0.025 wt% on the basis of the weight of the alcoholic solvent.

17. The method for manufacturing a slide structure having a liquid cover slip according to claim 11 , wherein the tissue slice used in the step (b) is a frozen section.