KR100536463B1 - Micro-dissection method - Google Patents

Abstract

The disclosed microdissection method comprises the steps of: (a) preparing a slide in which a tissue sample is wrapped with a liquid cover slip for enhancing resolution; And (b) observing and / or ablation of the slide using a correction unit capable of correcting the characteristics of the objective lens of the microdissection system.

Description

Micro-dissection method

The present invention relates to a microscopic ablation method, and more particularly, to a microscopic ablation method for observing tissues such as cells with an optical microscope, and collecting a predetermined region of the observed tissues by ablation using a laser.

In general, the microscopic ablation technique is to extract specific sites such as cells observed under an optical microscope for genetic changes or subsequent studies of DNA, RNA or proteins of specific cells or sites of biological tissues.

Prior art related to a conventional microdissection system is disclosed in US Patent Application No. 09 / 043,093, International Application Publication No. WO 99/39176, International Application Publication No. WO 00/34757, US Patent No. 6,010,888, and the like.

The conventional microscopic ablation technique is to dissect tissue using a laser, which is classified into two types according to the ablation method. One method uses the heat of the laser and the other method uses the laser as a knife.

Conventional microdissection methods include collection to collect the cells or regions to be excised, simply mount the slides, scan tissue samples placed on the slides in a predetermined path, or horizontally slide the slides to excise the cell samples. There is a need for a stage device for fine movement. The collector is typically installed in a collector holder or tray that is closely associated with the stage device. The collector holder and the stage device may have various structures according to the ablation method or the structure of the collector.

By the way, in most conventional microscopic ablation methods, the excised sample is collected in a cap rather than the main body of the collector. Therefore, the conventional microdissection method limits the slimming of the stage apparatus because a large amount of space is devoted to the collector body for mounting the collector body having a relatively longer length than the cap of the collector.

In addition, the conventional microablation method has a limitation in reducing the distance between the caps of the collection boxes because a plurality of collection boxes arranged in a row is employed in one system. On the other hand, in order to alleviate these problems, one holder may be used to mount several holders to a stage device using one holder, but such systems are also a limitation on slimming the collector holder and / or the stage device. The length of the path from the ablation zone to the corresponding collection bin is long enough to make the collection itself impossible or to increase the inefficiency of the collection.

On the other hand, when the space between the caps of the collection bin is farther away, it is relatively time consuming for the horizontal movement of the stage device and / or the tray (or stage) and the vertical movement of the stage device during a plurality of collection operations. Problems arise with efficiency.

In addition, the structure of the conventional holder holder has a problem that the task of attaching and detaching the collector to the holder is inconvenient, the conventional stage devices had a problem in stability and accuracy in the movement of the slide, in particular, the arrangement with the holder holder There have been many problems with the interrelationship and space efficiency of.

In general, the microscopic ablation technique requires not only optically precise observation of a tissue sample and a delicate selection of the area to be excised, but also a fine ablation of the selected sample using a laser, thereby making it possible to use slides used in conventional microscopes. Slide structure different from the structure is adopted. That is, the slide structure used in the microscopic ablation technique is a sample that is dyed and dried in a state in which the sample is simply placed on the slide without covering the sample with the cover glass or treating with the encapsulant.

Conventional fine ablation methods use the slide structure described above to observe and ablate a sample, so that fine ablation is performed in a state where the optical resolution of the sample is very low. Therefore, lesions with fine changes, such as precancerous lesions, may not be distinguished or identified to a sufficient extent in the microscope field of view.

In addition, known microscopic ablation methods seek to increase the optical resolution described above by using a precision objective lens. However, such a method increases the resolution of the objective lens as well as increases the price of the objective lens.

On the other hand, in the known fine ablation methods, the focal point for observation and the focal point for ablation are often different from each other, and thus it is necessary to compensate for the difference between these focal points. In particular, the need for such compensation increases when the spot size and focus of the laser beam are controlled according to the change of the objective lens and the state of the samples. However, the optical channels employed in conventional microdissection methods lack such compensation devices.

In the conventional microdissection method, the stage device can be operated only by a keyboard or a mouse connected with a computer. Accordingly, the necessity of a separate manipulator other than a keyboard or a mouse is increased for the convenience of the user in the operation of the microablation system.

In response to these demands, the proposed technique has been developed by Leica for manipulators that can operate X, Y and Z axes separately. The manipulator according to this technique has two rollers installed on one pillar to operate the X and Y axes of the stage apparatus, and a roller for operating the Z axis of the stage apparatus is installed on the other pillar.

Therefore, the Leica manipulator is expensive to manufacture the device, the volume of the device is not only large, there is a problem that the operation of the device is difficult.

On the other hand, conventional microscopic ablation methods are exquisitely excision of thin films or tissue sections using the photolysis principle of the laser. One of these methods utilizes a flattened thin film of polyethylene naphthalate (hereinafter also referred to as PEN) on a glass slide, glued along an edge. According to this technique, after placing a tissue sample on the center of the thin film of the PEN membrane and staining the tissue sample, a microscopic observation of the tissue sample selects a desired cell or cell population as a closed curve. The laser beam is precisely adjusted while irradiating the selected area along the closed curve. Excision is precisely performed by photolysis in the material layer irradiated with the laser beam, that is, in a PEN film including a tissue sample. When the first stage irradiation of the laser beam for ablation is terminated, after adjusting the depth of focus to a depth different from the focal point at the time of laser ablation, the second laser beam for irradiating the abrupt area from the remaining area is irradiated. The irradiation of the second laser beam adjusts the depth of focus so that the focus of the laser beam is not formed in the ablation region, unlike the depth of focus at the time of irradiation of the first laser beam. Since the laser beam irradiated in the second step is irradiated with an out-of-focus state, no further photolysis occurs in the ablation area, but a constant tension with respect to the area abated by the photon flow of the laser beam. ) Is going crazy. Therefore, when the area cut out by the tension of the laser beam is separated from the remaining area and moved upward, the collection of the cut out area is attached by attaching it to a collection tube coated with mineral oil at the top thereof. Is done. However, this method is so close that the distance between the focused and defocused areas for photocatalytic effects is so close that the partial or ablation zones are irradiated during the second stage laser beam irradiation. There is a disadvantage in that photolysis can occur.

The conventional microscopic ablation method developed to solve this problem is a tissue sample using a laser to solve the problem of partial photolysis during the second stage irradiation of the laser beam to deviate upwardly the tissue sample excised by the tension effect After ablation of the selected area, the ablation area was allowed to fall naturally, and a collecting tube was placed under the slide. However, the collection method by the natural dropping method of the restrained area has a problem that the collection rate is reduced due to the fine static electricity generated in the equipment.

All of the conventional microscopic ablation methods described above have a problem in that the optical resolution cannot be improved due to the limitation that the cover glass cannot be covered in the microablation process, and thus, fine observation and selection of the ablation zones are not easy.

The present invention has been made to overcome the above problems, 1) to improve the optical resolution of the tissue section when observed under a microscope to a sufficient extent, 2) to provide a laser beam and a correction unit generated by a laser device capable of energy and frequency adjustment It is an object of the present invention to provide a microdissection method that can easily excise tissue sections while maintaining an improved optical resolution using the microdissection system provided.

Another object of the present invention is to provide a microdissection method that can excise tissue sections over at least two times when the thickness of the cell tissue is thick.

Micro resection method according to a preferred embodiment of the present invention for achieving the above object, (a) preparing a slide in which the tissue sample is wrapped in a liquid cover slip for enhancing resolution; And (b) observing and / or ablation of the slide using a correction unit capable of correcting the characteristics of the objective lens of the microdissection system.

Preferably, the liquid cover slip is prepared based on a polyvinyl pyrrolidone polymer having a molecular weight of 10,000 to 360,000.

Preferably, step (a) maintains the height of the slide at approximately 14 to 26 μm; In step (b), the slide is excised at least twice at a predetermined depth.

Preferably, in the step (b), after cutting the closed curve of the ablation zone formed on the slide to a predetermined depth once with a laser device, the stage device of the fine ablation system is moved a predetermined distance in the Z direction before proceeding to the next turn. It comprises the step of.

Preferably, step (b) comprises: (b1) scanning a predetermined area of a live image of a tissue sample and storing it as a separate file; (b2) selecting an ablation zone in the file using only the file stored in the step; And (b3) matching the data value of the file obtained in step b3 to the live image of step (b1).

In the accompanying detailed description, reference is made to the accompanying drawings, which form a part, and is shown in a manner that describes specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims and their equivalents. In the description of the present invention, "vertical", "horizontal", "side", "up", "bottom", "rising", "falling", etc., are determined at the position of the microablation system in the context of the drawings and description. It should be regarded as a relative meaning to, and not as an absolute sense of.

The microdissection method according to the invention can in particular be used primarily for cancer research. In other words, the method can be used to determine the presence or absence of any specific gene at each stage of cancer development. In addition, the present method is to purely separate normal cells from tumor cells from target tissues in order to identify a tumor-specific genetic change at the DNA level, and by using the method of chemical photolysis without heating the biological cells 1 This is a method that can be cut at a resolution of 탆.

Hereinafter, with reference to the accompanying drawings will be described in detail a microdissection method according to a preferred embodiment of the present invention.

Since the microscopic ablation method according to the present invention observes and ablates the improved slide structure by mounting the improved microdissection system, a detailed ablation method will be described after describing the slide structure and the microdissection system.

The slide structure used in the preferred microdissection method of the present invention uses a polyvinyl pyrrolidone polymer. This polymer is a high molecular polymer material having various molecular weights, and is known to be harmless to the human body and to dissolve in both water and alcohol.

The resolution enhancing liquid cover slips surrounding the tissue samples are used by dissolving the polyvinyl pyrrolidone polymer using an alcohol solvent in a non-aqueous state. It is preferable to use an alcohol solvent, and more preferably ethanol or isopropanol, since internal components of the tissue sample are easily damaged by external air or moisture.

The slide structure has a coating layer for tissue sample protection. The coating layer may perform a protective film function to protect the tissue sample from external influences, as well as to improve the irregular morphology of the tissue sample. Therefore, when a tissue sample is observed using a microdissection system, it is possible to prevent diffuse reflection or scattering of light, and the refractive index of the polyvinyl pyrrolidone itself (1.43) is not significantly different from that of the tissue section. Because of this, high resolution is guaranteed.

Specific examples of the coating composition used to prepare the liquid cover slip for enhancing the resolution, the polyvinyl pyrrolidone polymer and UV laser beam having a molecular weight of 40,000 of 10% by weight relative to the weight of the ethanol solvent based on ethanol as a solvent A slide structure is prepared by including Congo Red, which contains 0.002 wt% based on the alcohol solvent reference weight as an absorption enhancer.

1 and 2 are cross-sectional views of the slide structure used in the fine ablation method according to a preferred embodiment of the present invention.

Referring to FIG. 1, a polymer film layer 172 is attached to an upper portion of a slide substrate 170 having a flat plate structure serving as a support layer by an interlayer adhesive member (not shown). The tissue sample 174 and the liquid cover slip 176 are stacked on the slide substrate 170.

Referring to FIG. 2, a slide substrate 180 having a window includes a polymer film layer 182 attached to an edge upper surface by an interlayer adhesive member (not shown), and a central upper surface of the polymer film layer 182. And a liquid sample slip 186 that completely encloses the tissue section.

The liquid cover slip 186 firmly adheres to the tissue sample 184 and at the same time completely surrounds the outside thereof, has a refractive index similar to that of the tissue sample, and has a flat interface with air. Therefore, by preventing the optical reflection or scattering during the observation using an optical microscope, and by improving the surface shape of the tissue sample, sufficient optical resolution during the observation of the microscopic area is maintained.

In addition, the liquid cover slip 186 prevents macromolecules, such as DNA, RNA, various proteins, etc., in the tissue sample 184 from being damaged or lost over time, and is excised from the tissue sample or DNA extracted from them. , Reliability of subsequent studies involving RNA and proteins.

In the slide substrate 180, a lower surface of the polymer film layer 182 on which the tissue sample 184 is seated is provided with a window for directly exposing the outside air. The window of the slide substrate 180 has a low temperature due to convection and conduction when the tissue sample 184, especially the frozen section, is mounted on the polymer film layer 182 while the polymer film layer 182 is in contact with the cryosection in the cryogenic state. In order to prevent the deterioration of adhesion of the frozen slices to the polymer film layer 182 due to the synchronization phenomenon, the attempt to mount the frozen slices while the window lead 188 is coupled to the window of the slide substrate 180 is performed. It is preferable. The direction in which the window lead 188 is detached from the slide substrate 180 is indicated by an arrow A. FIG.

The thickness of the slide structure shown in Figures 1 and 2 shows a marked difference from the actual, it should be fully understood that the thickness of each layer is slightly exaggerated for ease of explanation on the vertical structure, It is obvious that it is not illustrated in consideration of scale. The polymer film layers 172 and 182 are formed to a thickness of 1 to 2 micrometers (μm), and the tissue samples 174 and 184 are formed to a thickness of 2 to 6 micrometers (μm), the liquid phase according to the present invention. Cover slips 176 and 186 are typically formed to a thickness of 8 to 22 micrometers (μm). In particular, compared to the actual thickness of the slide substrate (170, 180), the thickness of the polymer film layer, the tissue sample and the liquid cover slip with the naked eye has a fine thickness that is difficult to display.

3 is a cross-sectional view schematically illustrating a fine ablation and collection operation for the slide structure of FIG. 2.

Referring to FIG. 3, in the state where the slide structure of FIG. 2 is mounted on the stage 190 of the microdissection system with the vertical direction reversed, the tissue sample 184 is moved in the direction of the arrow A using the observation unit. Observe. Subsequently, after selecting an ablation region (not shown), the polymer film layer 182 and the tissue sample in the region selected by the laser beam irradiated in the direction of arrow B from the upper portion of the slide structure in the inverted state. Both 184 and liquid cover slip 186 are ablation. Tissue sections to be excised are collected in a collecting container 192 disposed directly below it in the direction of arrow C under the influence of gravity.

On the other hand, it is not desirable to rely solely on how the excised tissue is collected by dropping by a simple gravity factor and collecting. Therefore, when the anti-static agent is included in the window slide substrate, the micro electrostatic force generated inside the slide structure can be removed, thereby improving the collection rate.

4 is a schematic structural diagram of a fine ablation system used in a fine ablation method according to a preferred embodiment of the present invention.

Referring to FIG. 4, the microscopic ablation system 1000 includes a first optical channel 10 including a CCD camera 34 for observing a sample (not shown) on a slide 40, and a selected predetermined region of the sample. Fixing the optical channel 100 including the second optical channel 20 including the laser device 22 and the like, and the slide 40 including the sample and fixing the slide 40 to X, Y. And a stage apparatus 200 for moving in the Z direction, a collector stage 250 for moving the collector holder 80 (FIG. 2) provided with the collector 70 relative to the stage apparatus 200, and To control the horizontal or vertical movement of the stage device 200 or to control the laser device 22, the objective lens 18, the collimation lens 21, the lamp of the microscope device, the lens, etc. of the optical channel 100. Control unit 300 to display a predetermined area of the sample observed by the optical channel 10 A display unit 400 including a monitor 402 or the like, and an operation unit 500 including a keyboard 502, a mouse (not shown), a manipulator 504, and the like, for operating the units. .

The microdissection system includes an improved optical channel 100, an improved stage device 200, and a manipulator 504 that can operate the stage device 200 independently of a mouse or keyboard. The optical channel 100 includes the correction unit 60 having a function of increasing the resolution of the fine ablation system during observation and ablation of a sample by supplementing the characteristics of the objective lens 18, and the stage apparatus 200. Has a holder holder and a collector stage to make the system slim and compact.

The control unit 300 performs bidirectional communication with the system hardware control unit, the system hardware control unit for controlling the system control software, the laser unit, the stage unit, the computer hardware, the CCD camera, the mouse, the manipulator, and the like, which collectively controls the entire system. And a laser focus controller, a laser power controller, a laser beam switch, a stage driver, and a collector drive unit, each of which performs a given function.

5 is a perspective view illustrating main parts of the stage apparatus 200 shown in FIG. 4, FIG. 6 is a plan view of FIG. 5, FIG. 7 is a right side view of FIG. 5, and FIG. 8 is of FIG. 5. It is a front configuration diagram.

5 to 8, the stage device 200 includes a base 210 having a first hole 212 formed therein and movable in a Z direction with respect to the frame 92 of the microscope device of the microdissection system 1000. ), The Y stage 220 provided in the base 210 so that the second hole 222 is formed and reciprocated in the Y direction, and the Y stage so that the third hole 232 is formed and reciprocated in the X direction. Collector stage 250 for reciprocating the X stage 230 installed in the 220 and the holder holder 80 placed in the holder insert 240 provided between the X stage 230 and the Y stage 220. ).

The base 210 has a mounting part 214 installed in the micro ablation system 1000 for vertical reciprocating movement, and a bracket 216 on which the collecting stage 250 is installed. A condenser is installed below the base 210.

The mounting portion 214 is coupled to the frame 92 of the microscope device by a slider device (not shown) used to move a sample in a conventional microscope device. The bracket 216 is for installing a collector stepping motor assembly for reciprocating the collector stage 250 in the X direction.

In the mounting portion 214 of the base 210, a Z shaft (not shown) used for the base 210 of a general microscope device is installed therethrough, and one end of the Z shaft is a Z stepping motor 218 installed in the frame 92. (See Fig. 1). The other end of the Z shaft is provided with a Z knob 211 that can manually raise and lower the base 210. The Z knob is similar to that of a conventional microscope device. The Z stepping motor 218 is responsible for the focusing of the first optical channel 10 for sample observation of the microablation system 1000 and the laser focusing function of 20 of the second optical channel for ablation of the selected sample region. do.

The first hole 212 penetrates the base 210 so as to have a size that does not interfere with the optical axis of the microablation system 1000 even when the X and Y stages 210 and 220 are moved to the maximum and minimum. Is formed.

The Y stage 220 is for moving the slide 40 in the Y direction for observation and ablation of the cells on the slide 40.

The Y stage 220 may have a substantially rectangular shape having a second hole 222 penetrated in the center thereof. The Y stage 220 is coupled to a Y ball screw 226 operated by a Y stepping motor 224 installed in the Y stepping motor mounting portion of the base 210. In order for the Y stage 220 to reciprocate in the Y direction from the top of the base 210, one side of the Y stage 220 is coupled to the Y ball screw 226, the Y guide on both sides of the base 210 Rails 228 are installed, and Y guide grooves 221 coupled to the Y guide rails 228 are formed at both bottom surfaces of the Y stage 220. Of course, the Y guide rail 228 and the Y guide groove 221 may be provided in the Y stage 220 and the base 210, respectively.

The second hole 222 formed in the Y stage 220 may have a fine ablation system 1000 even if the Y stage 220 moves to the maximum or the X stage 230 moves to the maximum and minimum with respect to the base 210. It is desirable to have a size such that it does not cause interference to the optical axis of.

The upper surface of the Y stage 220 is preferably a portion of the holder inserting portion 240 into which the holder holder 80 of the collector stage 250 can be inserted. Of course, when the holder insertion unit 240 is formed at a sufficient height on the lower surface of the X stage 230 to be described later, the upper surface of the Y stage 220 may be kept flat.

One side of the Y stage 220 is preferably provided with an X stepping motor installation unit on which the X stepping motor 234 may be installed.

The X stage 230 basically stores the slide 40 and simultaneously moves the slide 40 in the X direction for observation and ablation of cells on the slide 40.

Preferably, the X stage 230 has a substantially rectangular shape in which a third hole 232 is formed at the center thereof. The X stage 230 is coupled to an X ball screw 236 operated by an X stepping motor 234 installed in the Y stage 220. In order for the X stage 230 to reciprocate in the X direction from the top of the Y stage 220, one side of the X stage 230 is coupled to the X ball screw 236, the upper surface of both sides of the Y stage 220 The X guide rails 238 are installed, and X guide grooves 231 coupled to the X guide rails 238 are formed at both bottom surfaces of the X stage 230. Of course, the X guide rail 238 and the X guide groove 231 may be provided in the X stage 230 and the Y stage 220, respectively.

The third hole 232 formed in the X stage 230 may move the maximum X stage 230 with respect to the Y stage 220 or the maximum maximum and minimum Y stage 220 with respect to the base 210. Even if the size of the fine ablation system 1000 does not interfere with the optical axis.

A portion of the holder inserting portion 240 into which the holder holder 80 of the collector stage 250 may be inserted is formed on the bottom surface of the X stage 230. Of course, when the holder insertion unit 240 is formed at a sufficient height on the upper surface of the above-described Y stage 220, the lower surface of the X stage 230 may be kept flat.

The X stage 230 is provided with a member for fixing the position of the slide 40 across the third hole 232. To this end, one end of the third hole 232 is provided with an accommodating groove 233 into which one end of the slide 40 can be inserted and received, and the other end of the X hole may be selectively contacted with the slide 40. A slide retainer 235 is installed to slide on the stage 230.

The slide retainer 235 includes a retainer plate 235a inserted into a retainer groove 237 formed at one side of the X stage 230, and a retainer knob 236b installed through the retainer plate 235a. The retainer plate 235a has a position fixing groove 235c that can be engaged with one side of the slide 40, preferably the corner of the slide 40.

The collector stage 250 is for reciprocating the collector holder 80 located in the holder inserter 240 in the X direction. The collector holder 80 is installed so that the plurality of collectors 70 are compact and slim, which will be described later.

The collector stage 250 is moved by a collector stepping motor 252 installed on the base 210. To this end, a pinion 254 is installed on the shaft of the collector stepping motor 252, which pinion 254 is coupled with a rack 256 installed in the body of the collector stage 250. Meanwhile, a collector guide rail 258 is installed at the base 210, and a collector guide groove 251 is formed at the collector stage 250 main body to be fitted to the collector guide rail 258. The end of the main body of the collector stage 250 is provided with a connector 253 to which the coupling portion 94 of the collector holder 80 can be coupled.

The X, Y, Z, and Collector Stepping Motors 214, 224, 234, 252 are each 2 mm lead / precision grade ground to have specifications of 25000 to 50000 step / rev (6 nm / step). A lead / precision grade ground ball-screw is used. In addition, the X, Y, Z, and collector box stepping motors 214, 224, 234, 252 are each managed by a pair of optical limit switches 94. The accuracy of the position control of the X, Y, Z, and collector box stepping motors 214, 224, 234, 252 is approximately ± 8 μm, and the repeatability is approximately ± 0.5 μm. The travel speed and travel acceleration generated by the X, Y, Z, and collector box stepping motors 214, 224, 234, 252 are 20000-40000 step / sec and 20000-96000 step / sec2, respectively. .

The travel intervals of the X, Y, and collector stages 214, 224, 234, 252 are preferably 60 mm, 40 mm, 75 mm, respectively.

9 is a plan view of the holder holder, Figure 10 is a perspective view showing a state in which the collector is coupled to the holder holder.

9 and 10, in the microdissection system 1000 according to a preferred embodiment of the present invention, the collector holder 80 has a tubular collector body having a predetermined length with one end closed and the other open. 72, a collector cap 74 capable of closing the open end of the collector body 72, and a connecting ring 76 connecting the collector body 72 and the collector cap 74; It is for storing a plurality of collection boxes (70) compactly.

The holder holder 80 is a plate so as to communicate with the main body mounting portion 84 provided on both sides of the plate, an inlet portion 86 formed in the width direction center of the plate from the side of the plate, and the inlet portion 86 It is provided with an insertion portion 88 formed through.

The main body mounting portion 84 is disposed sequentially on one side of the plate to collect the first and second mounting portions (84a) (84b) and the other side of the plate, each having an elastic piece that can be elastically coupled to the main body The collector body 72 may be arranged in sequence so as to be elastically coupled, and the first and second installation units 84a and 84b may be provided with third and fourth installation units 84c and 84d disposed in a zigzag form. do.

The inlet 86 is preferably formed so as to penetrate in the width direction from the side of the plate so that the connecting ring 76 of the collecting box 70 does not interfere. The inlet 86 is for preventing the connecting ring 76 from interfering with the plate when the collection box 70 is mounted or detached to the collection holder 80. The inlet portions 86 are sequentially formed to correspond to the first to fourth installation portions 84a, 84b, 84c and 84d, and are arranged in a zigzag shape to form the first to fourth inlets 86a and 86b. 86c and 86d.

The inserting portion 88 has a shape in which the collecting cap 74 is elastically inserted to be fixed in position, and the first to fourth insertion holes 88a disposed in close contact with each other at minimum intervals in the longitudinal direction of the plate ( 88b) 88c and 88d.

Coupling portion 82 provided at the other end of the plate is not provided with the collecting box 70 is coupled to the connector 253 of the collecting stage 250 described above, the coupling guide 82a and the fixed plate A fixing knob 82b for fixing the position to the connector 253 is provided.

11 and 12, the holder holder 180 is for compactly storing a plurality of holders 70 in the same manner as the holder holder 80 described above.

The holder holder 180 is in communication with the main body mounting portion 184 provided on both sides of the plate, the inlet portion 186 formed in the width direction center of the plate from the side of the plate, and the inlet portion 186 The insertion part 188 is formed through.

The main body mounting portion 184 is the same as the main body mounting portion 184 of the above-described embodiment.

Unlike the inlet portion 86 of the above-described embodiment, the inlet portion 186 does not penetrate in the width direction of the plate, and an inlet groove is formed to have a predetermined depth of the plate. The retracting groove is provided with a connecting ring 76 of the collecting box 70 to prevent the connecting ring 76 from interfering with the other side of the plate.

The insertion part 188 has an insertion slit provided at the edge of the first through fourth insertion holes 188a, 188b, 188c and 188d so that the cap protrusion provided on the side of the collecting cap 74 is fitted. do. That is, the collection caps 74 are positioned in the insertion holes 188a-188d as a whole, and have a configuration in which the cap projection is fitted into the insertion slit.

Referring to FIG. 13, the optical channel 100 employed in the microablation system 1000 includes a first optical channel 10 and a second optical channel 20. The first optical channel 10 forms a first optical axis 12 of a microscope device (not shown), and the second optical channel 20 is a second optical axis of the laser beam generated from the laser device 22 ( 24).

The first optical channel 10 is an optical of a conventional microscope device capable of observing a sample (not shown) on the slide 40 using the eyepiece 32 or the CCD camera 34 of the observation unit 30. As a channel, it is formed by the combination of the eyepiece prism 14, the tube lens 16, the objective lens 18, the capacitor 11, and the lamp 13. The first optical axis 12 refers to an image observation path.

The second optical channel 20 is a movement path of the laser beam for cutting a predetermined region of the specimen, and the movement path is represented by the second optical axis 24. The second optical channel 20 has a prism 26, a pair of collimators 28, 21 and a shutter 23.

As shown in FIGS. 13 and 14, the second optical channel 20 has a substantially closed channel box 25 so that a predetermined optical axis can be stably formed even if the intensity of the laser beam is changed as necessary. It is preferably installed in.

The laser device 22 is designed to have a spot size of 1 μm or less, and the power of the laser beam generated therefrom is designed to be adjustable according to a user's request. The laser device 22 has a solid state laser with a wavelength of 355 nm, the specification having 20 uJ and a frequency of 50 to 10000 Hz.

One end of the channel box 25 is formed with a beam inlet 27 so that the laser beam generated from the laser device 22 can be incident. First and second ports 29a and 29b are provided at the other end of the channel box 25 so that the first optical axis 12 may be formed. The first port 29a is a portion connected to the observation unit 30, and the second port 29b is a portion connected to the objective lens 18.

Referring to FIGS. 13 and 14, the prism 26 is capable of refracting the laser beam generated from the laser device 22, such as an ultraviolet laser, in the direction of the collimator 21, 28. ) Is installed inside.

The pair of collimators 21 and 28 has the function of helping the microablation system to achieve a predetermined spot size by ultimately forming the laser beams passing through the prism 26 in parallel. In other words, if the collimators 21 and 28 are incorrectly determined or arranged, the system may not only obtain parallel laser beams, but may also result in inadequate focus even if it is impossible to focus or close. The collimator is divided into an eyepiece collimator 28 in the form of a concave lens and an objective collimator 21 in the form of a convex lens.

The collimators 21 and 28 are respectively installed at one end of the barrel having a predetermined length. The eyepiece collimator 28 has a concave lens shape in which both surfaces are substantially symmetrical. The object collimator 21 is arranged so that the concave lens 21a provided to face the shutter 23 and the convex lens 21b provided to face the dichroic mirror 50 to be described later are adjacent to each other, and the concave lens 21a is disposed. When the convex lens 21b is arranged as a whole, it is preferable to exhibit the characteristics of the convex lens.

The shutter 23 is installed between the eyepiece collimator 28 and the objective collimator 21, it is preferable to have a structure that is opened when cutting the sample, otherwise closed.

The dichroic mirror 50 is disposed on the first optical axis 10 in the vicinity where the second optical axis 20 is refracted. The dichroic mirror 50 passes the first optical axis 10 in a straight line and refracts the second optical axis 20. That is, the dichroic mirror 50 transmits the image of the sample observed through the objective lens 18 of the microscope device to the observation unit 30 side, and transmits the laser beam incident from the laser device 22 to the objective lens 18. In the direction of. The dichroic mirror 50 is a flat mirror coated with a transparent multilayer thin film. When the incident angle of light is 45 °, light of a certain wavelength range is reflected by the interference effect of light in the thin film, and the other is It has the property of permeation. The dichroic mirror 50 can have a flat plate shape and a prism shape, but in this embodiment, a flat plate shape is used. Of course, the thickness or the number of layers of the film can be adjusted, and part of the visible light can be freely selected and used depending on the material.

13 and 14, a correction unit 60 having a concave lens characteristic is disposed between the dichroic mirror 50 and the observation unit 30, and between the correction unit 60 and the dichroic mirror 50. The filter 70 is installed.

The correction unit 60 has a function of increasing the resolution of the fine ablation system during observation and ablation of the sample by supplementing the characteristics of the objective lens 18. In addition, the correction unit 60 is for correcting the difference between the focus of the sample recognized by the CCD camera 34 or the eyepiece 32 and the focus when the predetermined area of the sample is ablated using the laser beam. . By this correction unit 60, the spot size of the laser beam of the fine ablation system can be kept constant regardless of the power change of the laser beam. In particular, even when the laser beam power is increased to speed up the ablation operation, the spot size of the laser beam can be kept constant by the correction unit 60.

The filter 70 blocks the heat of reflection of the laser beam passing through the dichroic mirror 50 and ultimately prevents the observation unit 30, that is, the eyepiece 32 and the CCD camera 34 from being damaged. Has The filter 70 uses a flat mirror or plastic, and is disposed to be inclined at a predetermined angle with the first optical axis 10.

It is preferable that the correction unit 60 uses a concave correction lens having a characteristic capable of correcting the resolution in response to characteristics such as magnification of the objective lens 18 of the microscope device. The correcting lens 62 of the correcting unit 60 is axially aligned with the first optical axis 10. The correction lens 62 has a flat portion facing the dichroic mirror 50 and a portion facing the observation unit 30 has a concave shape.

16 and 17, in the optical channel, the correction unit 60 includes a plurality of correction lenses capable of correcting each objective lens according to the characteristics of the plurality of objective lenses installed in the microscope device. 62 is provided. Each of the correction lenses 62 needs to be aligned with the first optical axis 10 so as to correspond to the corresponding objective lens. To this end, the correction unit 60 includes a correction tray 64 in which a plurality of correction lenses 62 are radially installed and rotatably installed at a predetermined angle.

The correction tray 64 is installed on the rotation shaft 66 rotatably installed in the channel box 25. The gear 64a is formed in the outer peripheral surface of the correction tray 64, and this gear 64a meshes with the gear 68a provided in the rotating shaft of the motor 68. As shown in FIG. Of course, the correction tray may be configured to be directly connected to the rotating shaft of the motor. The motor 68 is controlled by a control unit, not shown, and is capable of forward and reverse rotation.

Six correction lenses 62 are arranged at the 60 degree angle in the correction tray 64. Therefore, in the fine ablation system, when the magnification change is generated by the change of the objective lenses, the correction lens 62 corresponding to the objective lens is rotated by the rotation of the correction tray 64 rotated by the motor 68. It can be located on the optical axis 10, so that the correction lens 62 can correct the objective lens 18 during operation of the system.

FIG. 18 is a perspective view schematically illustrating the manipulator shown in FIG. 4, and FIG. 19 is a partial cutaway view of FIG. 18.

As shown in FIGS. 18 and 19, the manipulator 504 includes a pad 510 that can be placed on a desk (not shown), a hollow pole 520 provided on the pad 510, and a pillar. Three rolls 530 installed on the outer circumferential surface of the 520 and the rotation of the sensing member 540 and the rolls 530 installed on the pillar 520 to detect the amount of rotation of the respective rolls 530. The control member 550 installed in the pad 510, the separator 560 separating the some rolls 530 from each other, and the cap 570 provided at the end of the pillar 520 so as to control the movement of the stage apparatus according to the quantity. ).

The pad 510 has a flat case structure in which a control member 550 connected to power applied from the outside is embedded. The pad 510 is provided on the upper surface of the pillar 520 installation hole 512 in which the pillar 520 may be installed. The top cover of the pad 510 is provided with lamps 514 for displaying the power on, off and various operating states, and buttons 516 for other operations of the stage apparatus. The lamps 514 and the buttons 516 are driven by the control member 550 installed inside the pad 510.

The pillar 520 is installed on the pad 510 and protrudes around the insertion portion 522 and the insertion portion 522 inserted into the installation hole 512 of the pad 510 so as to pad the pillar 520 to the pad 510. ) Is provided with a support portion 524. Screw support holes 526 are formed in the support part 524. The screw coupling hole 526 is for fixing the pillar 520 to the pad 510 by a bracket 528 positioned below the top cover of the pad 510.

The rolls 530 are provided with a Z-axis roll 530a, a Y-axis roll 530b, and an X-axis roll 530c in order from the top to the bottom of the drawing, respectively. Of course, the order may be modified as many as necessary.

The rolls 530 are installed to rotate around the column 520 by bearings 532, respectively. Each of the rolls 530 is rotated independently of each other by being positioned at the bottom of the bearing 532 and separated from each other by the support 534 fixed to the pillar 520. On the outer circumferential surface of each roll 530, slits of a predetermined pattern are formed to facilitate rotation of the roll 530. On the inner circumferential surface of each roll 530 the inner wall of the roll 530 so that the inlet 536 and the bearing 532 introduced into the inner wall of the roll 530 may be installed so that the modulator 542 to be described later is installed. And a bearing mounting portion 538 protruding therefrom. Reference numeral 531 in the drawing represents a spacer for the installation space of the separator 560.

The sensing member 540 includes a modulator 542 fixed to an inlet 536 provided on an inner wall of the corresponding roll 530, and a circuit board installed on an inner wall of the pillar 520 so as to be connected to the control member 550. 544 and a pair of photointerrupters 546 electrically connected to the circuit board 544 so as to be disposed on both sides of the modulator 542, respectively.

The sensing member 540 detects whether light is blocked by interference of the modulator 542 and generates a signal for controlling coordinates of the stage device according to the waveform.

As shown in FIG. 20, the modulator 542 is a type of encoder, and each modulator 542 is preferably annular. The outer circumferential surface of each modulator 542 is fixed to the inlet portion 536 of the corresponding roll 530, and a through hole 542b is formed at the center of the modulator 542, which is located close to the outer circumferential surface of the pillar 520. . The body of the modulator 542 is provided with a plurality of slit holes 542a formed at radial equal intervals with respect to the center line of the pillar 520. In the optical interference of a pair of photo interrupters 546 adjacent to each other, the slit holes 542a are preferably spaced apart from each other so as to maintain a phase difference of 90 degrees. In other words, the slit hole 542a of the modulator 542 rotates between the pair of photo interrupters 546 while the adjacent photo interrupters 546 alternately interfere with light while maintaining a phase difference of 90 degrees.

As shown in FIG. 21, each photointerrupter 546 includes a light emitting unit 546a such as a light diode capable of emitting light such as an infrared frequency and a photo transistor for receiving an infrared frequency. and a light receiving portion 546b such as a transistor). Reference numeral 546c in FIG. 16 denotes a connection portion electrically connected to the circuit board 544, and reference numeral 546d is a space in which the modulator 542 can be located.

When the light passes through the slit hole 542a, the photo interrupter 546 does not generate light interference. When the light does not pass through the plate between the slit holes 542a, light interference occurs, thereby causing a modulator ( According to the rotation operation of the 542, the waveform of the signal received by the light receiver 546b may be digitized. The pair of light emitting portions 546a of the photo interrupter 546 are arranged to have a phase difference of 90 degrees with each other.

On the other hand, the sensing member may be of any known technology similar to that can be employed to sense the rotation amount of the roll in addition to the optical interference technology such as the modulator and the photo interrupter.

18 and 19, the circuit board 544 is used to control the photo interrupter 546, and an amplifier for amplifying a current impulse value due to the rotation of the modulator 542 into which the light receiving unit 546b flows. (Not shown) and the like are arranged.

The control member 550 determines the sum of the current and its current impulse values and the type of the current by the rotation of the modulator 542, and transmits these values to the stage controller of the control unit (not shown) through the RS-232 interface. Send or control the stage device directly. That is, the current impulse increases or decreases by the rotation of the modulator 542, and the coordinates of the stage device can be moved when the value of the impulse is determined by the increase or decrease.

On the other hand, the control member 550 may set different coordinate movement displacements of the stage apparatus per unit rotation of the roll (modulator) according to the request of the consumer through a setting interface.

The separator 560 is for distinguishing the Z-axis roll 530a and the Y- and X-axis rolls 530b and 53c, and one end thereof is installed between the Z-axis roll 530a and the Y-axis roll 530b. The other end is installed in the pad 510.

The separator 560 is bent from the inclined plate 562 to be arranged in parallel with the bottom surface of the inclined plate 562 and the Z-axis roll 530a provided at the position where the X, Y axis rolls 530b and 530c are positioned. A flat plate 564 is provided. The shape of the separator 560 is ergonomically designed to facilitate the user's finger movement and to distinguish the vertical plane from the X and Y planes when the coordinates of the stage device are converted.

The cap 570 is preferably installed at the end of the pillar 520 in the interference fit method. The diameter of the cap 570 is made substantially the same as that of the roll.

A method of fine ablation according to a preferred embodiment of the present invention using the slide structure and the micro ablation system described with reference to FIGS. 1 to 21 will now be described in detail.

The fine ablation method according to the preferred embodiment of the present invention may store not only a real-time image mode expressed in the display unit, but also a pre-scanned image or, if necessary, immediately search for an image region of interest using a roadmap image, and search for the image region of interest. By selecting an ablation zone for the captured image and storing such data values, tele-consultation is possible through on-line or the like.

First, the power of the system shown in FIG. 1 is turned on, and a program for operating a fine ablation system on a computer screen is operated. Subsequently, a laser key (not shown) installed in the computer is rotated to turn on the laser device managed separately. Next, the laser is turned on on the dedicated program. The laser then starts to warm up.

Subsequently, the above-described slide structure is fixed to the receiving groove of the X stage 230 of the stage apparatus for the microdissection system 1000 in order to observe and ablate the cell region using the microdissection system 1000. Then, the stage device 250 finds the absolute coordinates of the slide 40, sets the slide to the initial position, collects the collector holder 80 having the collectors 70 installed therein, and connects the collector stage 250 to the collector. The holder 80 is also set to the initial position. In this state, the objective lens is set at the smallest magnification (4 ×).

Then, the control unit expresses the cell tissue on the slide in the form of a live image on the screen through the CCD camera. In this process, the control unit adjusts the above-described axis by dividing the vertical and horizontal directions on the screen into two parts, respectively, in order to adjust the axis of the live image of the cell tissue displayed on the screen through the slide. In this case, the live image on the screen is automatically turned off, that is, 'camera off'.

Subsequently, the user may adjust the maximum resolution of the CCD camera, the magnification of the objective lens, etc., but autofocus the cell tissue by the control unit autofocus function. This auto focusing is possible by the fine movement of the base operated by the Z stepping motor of the stage device 200. In addition, the user may observe the cell tissue while moving the stage device 200 by using the operation unit 500 such as a keyboard, a mouse, and a man plate 504. Moving the stage device 200 for observation of the cells on the slide 40 using the microdissection system 1000 primarily operates the X stage 230 and the Y stage 220 in the X and Y directions, respectively. As the X and Y stages 210 and 220 are driven independently, the X and Y stages 210 and 220 may be operated simultaneously to move the X and Y stages 210 and 220 simultaneously.

The movement of the Y stage 220 relative to the base 210 causes the X stage 230 to also move in the Y direction, and as a result, the slide 40 moves. Then, the observation area displayed on the monitor through the optical channel 100 of the microablation system 1000 is also moved. However, the movement of the X stage 230 relative to the Y stage 220 only moves the X stage 230 itself. Moving the stage device for observation of cellular tissues is only possible when in the 'move' command mode of the program.

Although the user may simply observe the cell tissue through the screen and the alternative lens, the work is often performed to remove a portion of the cell tissue placed on the slide. Such ablation can be performed on a live image, but typically, a roadmap function is used to store the shape of cell tissues on a slide in a computer, and then perform necessary tasks.

For the road map function, when the user clicks on the road map icon on the program, the control unit automatically sets the objective lens to 4 magnifications, and then reads all areas of the slide into predetermined frames (12 x 8 frames). Begin to enter. This process is displayed on the top screen of a separate stage control box in the program, and saved as a jpg file for each frame, and the control unit combines the image of the local area for each frame obtained by the CCD camera to create a mosaic-type overall image. It is displayed on the top screen of the stage control box. The screen then displays an image of the cell tissue of the portion located on the optical axis on the slide. In addition, the overall image is made such that the entire image is connected without interruption on a reduced scale. The reduced accumulation is automatically calculated so that the overall image is displayed properly on the monitor screen. That is, the user can open a search file and bring up a screen by using a mouse. At this time, the user may select only a part of the screen for detailed observation. The selected part is displayed as a navigation picture in the view window, which is automatically calculated according to the size of the computer monitor. A specific position of the view window, for example, an upper left corner point, may be set as a starting point for the search module for the overall image.

The search process for the general image uses a zoom function for detailed observation of the cell image. The picture file number of the image selected by the user is determined according to the view window coordinates indicated in the mosaic image construction process.

Meanwhile, the image may be moved in a desired direction by dragging the image through a mouse operation, or the image may be moved in a desired direction by manipulating the manipulator. This moving operation is possible by the X and Y stages of the stage device.

In addition, when the user clicks the memory button on the program, the user can conveniently operate the system in subsequent observation and ablation operations by storing an optimal focusing state for each magnification in observing the cell tissue controlled by the control unit. have. On the other hand, the user can mark any point on the cell tissue that the user needs, that is, the area to be observed or ablation, by using the marking function on the program. This marking function is a function that allows the control unit to automatically find the coordinates of such marking by double-clicking the marking on the program with a mouse. The marking function is possible in the ablation mode as well as the observation mode.

The user may then scan the required area on the slide. When the scanning button of the program is pressed for scanning, the control unit allows the user to select the upper right margin and the lower left margin of the area to be scanned on the screen. When selected by the user to the scanning button, the control unit turns off the live image on the screen. This scanning function is not performed at the minimum magnification, but is related to observation and ablation of the cell area of interest, so scanning is performed using the selected objective lens, and a predetermined frame (width × length: 4 × 4) is performed. Is scanned. The scanned file is automatically saved on your computer. The scanned file can exchange its data values among a plurality of users online, and the file scanned by a third party can be used for drawing operations described below by using a conventional application without using a microscope device or a microablation system. can do. However, it is not possible to perform an ablation operation only with the scanned file.

On the other hand, when the scanning operation is completed for each frame by the scanning command, the live image disappears and the scanned image is displayed on the computer screen. However, in this state, when the 'move' command is issued on the scanning control box, the screen moves. This is not because the stage device is moved but the conversion of data values. In addition, the scanned image also forms a comprehensive image in a mosaic form.

Next, an operation (drawing operation) of selecting an ablation region through an observation process, such as a live image itself observed through a CCD camera or a part of cell tissue stored by a road map function, will be described.

The drawing operation is mainly performed by drawing a closed curve on a computer screen by using a drag function of a mouse. The program also allows the user to select various modes, such as circles, ellipses, freehands, and the like. Regarding the drawing task, all that is required is to select a collection box from which the tissue to be excised can be collected. According to the ablation method according to the present embodiment, the program allows the user to decide to collect in advance. The control unit automatically assigns a color to indicate the cutting path according to the type of cell sample at the time of collection selection. When cell tissue is excised by the ablation operation described later, the excised tissue is collected in a preselected collection bin. In addition, the drawing path is displayed together with the cell image according to the accumulation selected by the user on the monitor screen, and the coordinates of the drawing path may be stored in the computer memory.

On the other hand, even if the user or a third party does not use the fine ablation system, as described above, it is possible to draw a required area using only the pre-scanned file.

Next, the operation of cutting the cell region determined by the drawing operation will be described. When the user issues an ablation command on the program, the microdissection system aligns the optical axis with the collection to which the tissue to be excised is to be collected. In this process, the control unit checks the position of the adjacent collection box with reference to the first collection box.

The ablation operation may also ablate individually selected drawing regions, but may also ablate a plurality of ablation portions sequentially. In this case, the order of ablation is in the selected collection order.

A specific ablation operation is made possible by the control of the control unit by cutting the path by means of a laser beam irradiated through the laser device while the stage device once cycles the drawing path along the coordinates of the drawing path selected by the user. Of course, in the ablation operation, the user can appropriately adjust the power of the laser beam.

The microscopic ablation method of the present invention uses a slide structure with a liquid cover slip in particular, and a so-called 'multi-layers cutting' concept is introduced for the case where the cells are thick such as plant cells.

In the multi-layer cutting operation, as shown in FIG. 22, the cutting operation is not performed while circulating the drawing area once but cutting the selected drawing area by circulating at least two times. That is, after first cutting (S10) to a predetermined depth while circulating the drawing path of the slide structure, using the Z stepping motor of the stage device, the stage device is moved by a predetermined interval in the Z direction to increase the height of the slide structure. Secondary ablation (S20) to a predetermined depth while circulating the same drawing path again, and finally, after raising the slide structure by a predetermined height, it means tertiary ablation (S30) while circulating the same drawing path. The multi-layer ablation can be adjusted in accordance with the thickness of the cell tissue.

On the other hand, in the microdissection method according to a preferred embodiment of the present invention, tissue sections collected by being excised from cell tissues and collected may be identified by a microdissection system. In other words, when the user presses the 'confirm collection' button on the program, the X stage on which the slide is installed is moved as much as possible in the direction of the + Y axis (frame) and the base is raised as much as possible. Then, the microdissection system can identify the tissues excised in the corresponding collector using the 4x objective lens. Auto focusing is possible during this verification.

In the fine ablation method according to an exemplary embodiment of the present invention, the ablation operation may be performed after a third party sets the scanning file instead of the live image and matches the drawing path to the actual live image.

To this end, the user loads a scanning file having a drawing path value in a program, and then mounts a slide structure corresponding to the scanning file in the microscopic ablation system. Subsequently, when the 'send to stage' button on the browser is pressed, all data values of the scanning file, which are processed by the third party, including the drawing path and the marking area, are displayed on the screen corresponding to the values of the live image.

In this work, the method introduced the concept of 'correlation tracking'. In other words, the above-described correlation tracking concept compensates for the error in coordinates generated while mounting the slide structure in the microscopic ablation system. This algorithm makes it possible to perform realistic ablation using the data values of the scanned image.

Experiments were conducted to confirm the improvement of the optical resolution and the preservation of the internal components in the tissue samples. Such improvement of the optical resolution of the present invention could be sufficiently confirmed through specific experimental data.

23 to 25 are comparative pictures according to a conventional comparative example and a preferred embodiment of the present invention for explaining the difference in optical resolution. Tissue samples observed in FIGS. 23 to 25 are image images observed with a microscopic ablation system according to the present invention with respect to tissue samples prepared for cryosection after being collected for observation of colorectal cancer, liver cancer and gastric cancer in clinical patients, respectively. .

23 to 25, the image photographs on the right of the center arrow in each of the drawings are photographs of an example of observing a tissue sample using a microdissection system according to a preferred embodiment of the present invention. Each image photograph is an image taken through a conventional slide structure and an optical microscope.

First, after a predetermined dyeing process for the tissue sample mounted on the upper slide, the tissue sample that is exposed to the outside air was observed with an optical microscope to obtain a comparative example photograph, after which the resolution according to the present invention After coating the tissue samples with the enhancement liquid cover slip, each tissue sample was observed again with a microablation system to obtain an example photograph.

As shown in FIGS. 23 to 25, the degree of improvement of the optical resolution may be intuitively confirmed. That is, in the photographs of Comparative Examples (1A of FIG. 23, 2A of FIG. 24, and 3A of FIG. 25), the surface of the stained tissue sample is not kept flat, so that light scattering occurs and the cells to be observed are black. It can be seen that it is difficult to identify between each cell. However, in the photographs according to the embodiment of the present invention, a clear distinction between cancer cells and inflammatory cells can be seen (FIG. 23B), and the cancer cells form a structure and clearly show the appearance (2B of FIG. 24). It can be seen that even the diffused appearance (3B of Figure 25) can be clearly seen. Therefore, it can be seen that the liquid cover slip according to the present invention serves as a functional layer to enhance the optical resolution of the tissue sample on the slide.

The fine ablation method according to the present invention has the following effects.

First, the optical resolution has been improved to enable fine observation of tissue samples and precise selection of microscopic areas.

Second, the optical resolution can be improved during ablation by the correction unit including a concave lens-type correcting lens capable of correcting the characteristics of the objective lens, thus enabling fine observation of tissue samples and precise ablation of fine areas. .

Third, by introducing a correction unit, the difference between the focus of the laser beam required for ablation of the sample and the focus required for observation of the sample can be corrected. Therefore, the ablation operation is maintained by keeping the spot size of the laser beam emitted from the laser device constant. Precision and stabilization can be expected.

Fourth, the plate of the collector holder can be made thin by ideally disposing the cap, the connecting ring and the body of the collector on the plate of the collector holder. Therefore, the space according to it can be minimized, and the collectors can be mounted stably in the collector holder, and the collection efficiency can be guaranteed.

Fifth, after transmitting to the third party an enlarged image image precisely observed due to the enhancement of macromolecular preservation in the tissue sample and the enhanced resolution upon optical observation, a sufficient and reliable opinion is received from the remote person. It is preferable because it can provide the basic technical base which can perform fine ablation.

Sixth, stabilization and perfection of the ablation work can be achieved by the multi-layer ablation concept, and the extent of ablation can be expanded according to the thickness of the tissue sections.

Seventh, since the stage device can be moved separately from the keyboard or mouse, the microablation system can be conveniently operated.

It is to be understood that the above detailed description is intended to be illustrative, and not restrictive. Many other embodiments will be apparent to those skilled in the art upon reading or understanding the above detailed description. It should be noted that the embodiments discussed in other parts of the detailed description and discussed with reference to other figures may be combined to form additional embodiments of the present invention. Accordingly, the scope of the invention should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

The following drawings attached to this specification are illustrative of preferred embodiments of the present invention, and together with the detailed description of the invention to serve to further understand the technical spirit of the present invention, the present invention is a matter described in such drawings It should not be construed as limited to.

1 and 2 are cross-sectional views of a slide structure with a liquid cover slip used in the microscopic ablation method according to a preferred embodiment of the present invention.

FIG. 3 is a cross-sectional view illustrating a fine cutting and collecting operation of the slide structure of FIG. 2.

4 is a schematic structural diagram of a fine ablation system used in a fine ablation method according to a preferred embodiment of the present invention.

FIG. 5 is a perspective view illustrating main parts of the stage apparatus shown in FIG. 4; FIG.

6 is a plan view of FIG. 5.

7 is a diagram illustrating the right side of FIG. 5.

8 is a front configuration diagram of FIG. 5.

Figure 9 is a plan view of the holder holder used in the microdissection method according to a preferred embodiment of the present invention.

10 is a perspective view showing a state in which the collector is coupled to the holder.

11 is a top view of another collection holder.

12 is a perspective view illustrating a state in which a collector is coupled to the holder of FIG. 11;

FIG. 13 is a schematic structural diagram of an optical channel of the microdissection system shown in FIG. 4. FIG.

FIG. 14 is a sectional view of a channel box in which the optical channel and correction unit part of FIG. 13 are installed; FIG.

FIG. 15 is a plan view of FIG. 14;

FIG. 16 is an enlarged plan view of a portion of the correction unit of FIGS. 14 and 15.

17 is a cross-sectional view taken along the line A-A of FIG.

18 is a perspective view schematically showing the manipulator shown in FIG.

19 is a partial cutaway view of FIG. 18.

20 is a plan view of the modulator shown in FIG. 18;

FIG. 21 is a side configuration diagram of the photo interrupter shown in FIG. 18. FIG.

22 is a conceptual diagram illustrating a fine ablation method according to a preferred embodiment of the present invention.

23 to 25 are comparative pictures according to a conventional comparative example and a preferred embodiment of the present invention for explaining the difference in optical resolution.

<Explanation of symbols for main parts of the drawings>

10 first optical channel 12 first optical axis 14 eyepiece prism

18 ... objective lens 20 ... second optical channel 22 ... laser unit

24 ... 2nd optical axis 25 ... Channel box 26 ... Prism

21, 28 ... collimator 30 ... observation unit 32 ... eyepiece

34 ... CCD camera 40 ... Slide 50 ... Differential mirror

60 ... calibration unit 62 ... compensation lens 64 ... compensation tray

66 ... rotation shaft 68 ... motor 70 ... collection box

72 ... collection body 74 ... collection cap 80 ... collection holder

82 Joining part 84 Main body mounting part 86

88 Insert ... 200 Stage unit 210 Base

220 ... Y stage 230 ... X stage 240 ... holder insert

250 Collector Stage 504 Manipulator 510 Pad

520 posts 530 rolls 540 sensing element

542 Modulator 544 Circuit Board 546 Photointerrupter

550 ... Control member 560 ... Separator 570 ... Cap

Claims (9)

 In observing a slide wrapped with a tissue sample with a resolution cover liquid enhancement slip with a calibration unit mounted optical channel and finely cutting it with a laser device, the liquid cover slip is mainly composed of a polyvinyl pyrrolidone polymer having a molecular weight of 10,000 to 360,000. After cutting the closed curve of the ablation zone formed on the slide to a predetermined depth with a laser device, the stage device of the microablation system is re-cutted by a predetermined interval in the Z direction before proceeding to the next turn, and the holder of the stage device is inserted. And a resected tissue sample collected in the collection container inserted into the reciprocating portion, the body disposed on the longitudinal side, and the cap inserted into the insertion hole. delete delete delete The method of claim 1, wherein the tissue sample scans a predetermined area of a live image and stores the data as a separate file, selects an ablation area in the stored file, and matches the data value to the live image. delete delete delete  According to claim 1, wherein the stage device is installed on the microscope frame of the optical channel to be reciprocating in the Z-axis direction, the base having a first hole through which the first and second optical axes pass, and the reciprocating movement in the Y-axis direction A Y stage provided at the base to form a second hole through which the optical axes pass, and an X stage formed at the Y stage to enable reciprocating movement in the X axis direction and a third hole formed to pass through the optical axes and installed in the slide. Fine ablation method to be equipped with.