KR20030042231A - Operation method of laser microdissection system - Google Patents

Abstract

PURPOSE: Provided is a fine laser cutting system in which absolute coordinate is established according to the physical zero point shown on a slide and selection of a cutting part and cutting operation are carried out in turn, eliminating the restriction of time and space in the process of part selection and operation of cutting. CONSTITUTION: The operation method comprises following steps of: loading a slide(70) on a stage equipment at a decided place; moving the stage equipment and deciding zero point(78) of cutting operation by recognizing zero point on the slide by picture; reading by a computer memory a coordinate value based on the zero point of the cutting part contained in the total picture of a tissue piece(74) attached to the slide; and cutting the cell by irradiating laser beam by moving the stage equipment according to the coordinate value of the cutting part based on the zero point.

Description

Operation method of laser microdissection system {OPERATION METHOD OF LASER MICRODISSECTION SYSTEM}

The present invention relates to a method of operating a laser microsurgery system, and more particularly, to a method of operating a laser microsurgery system capable of performing microsurgery not only in a real time image mode but also in a stored image mode.

In recent years, the main field of cancer research has been to find out which specific gene abnormalities are involved in each stage of cancer development. Pure separation of things should first be done. Therefore, in these studies, a sophisticated microdissection technique for selectively obtaining cells according to the stage of cancer development is absolutely required. Currently, several companies such as PALM (registered trademark) develop a microdissection system using a laser. It is actively marketed. This laser microdissection system is a device for microdissection by chemical photolysis without heating biological cells, and can cut cells at a resolution of 1 [mu] m.

However, since all conventional laser microdissection systems do not set the concept of absolute coordinates as a reference, there is a problem in that ablation can be performed only in a state in which tissues are photographed in real time, that is, under a real-time image mode. That is, according to all conventional laser microdissection systems, there is a spatial constraint that the selector of the ablation region stays in the physical space in which the microdissection system is installed, and performs ablation operation. There was a time constraint that it was difficult to perform.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-described problems, by constructing the absolute coordinate system by the physical zero displayed on the slide, and performing the ablation site selection operation and ablation operation based on the absolute coordinate system and The purpose of the present invention is to provide a method for operating a laser microdissection system that eliminates the time and space constraints between the ablation operations.

The operating method of the laser microdissection system of the present invention is mounted on the movable stage and the image recognition of the physically displayed zero point on the slide to which the tissue section is fixed is set to the zero point of the coordinate system, the ablation area displayed on the image captured tissue section The microscopic ablation is performed by moving the stage based on the coordinate value in a state in which a coordinate value based on the zero point is assigned to.

In the above-described configuration, it is preferable that the zero point is displayed at a portion which is not occupied by the tissue section on the slide.

1 is a schematic appearance configuration diagram of a laser microdissection system to which the present invention can be applied;

2 is an electrical functional block diagram of a laser microdissection system to which the present invention can be applied;

3 is a functional block diagram showing the operation method of the laser microdissection system of the present invention in units of modules;

Figure 4 schematically shows the cross-sectional structure of the slide that can be applied to the operating method of the laser microdissection system of the present invention,

5 is a flowchart illustrating a process of acquiring a comprehensive image of a scan area in a slide in the method of operating the laser microdissection system of the present invention;

6 is a view for explaining a process of displaying a zero point on a slide in the operating method of the laser microdissection system of the present invention,

7 is a view for explaining a process of setting a scan area and a local area in the operating method of the laser microdissection system of the present invention;

8 is a flowchart illustrating a process of performing a fine ablation operation on a slide in the method of operating the laser microablation system of the present invention.

*** Explanation of symbols for the main parts of the drawing ***

1: CCD camera, 2: alternative lens,

3: laser optical channel, 4: objective lens,

5: stage top plate, 5a: fixing member,

6: sample holder,

7: bottom stage, 8: condenser,

9: dimming filter, 10, 13: laser reflector,

11: laser focus lens, 12: laser condenser lens group,

14: laser generator, 15: cooling fan,

16: halogen lamp, 17: computer body,

18: monitor, 19: keyboard,

20: mouse, 21: laser / stage controller,

30: computer hardware, 31: system control software,

32: system hardware control, 33: mouse,

34: CCD camera, 35: laser focus control unit,

36: laser power control, 37: laser beam switch,

38: stage driving unit, 39: collection box driving unit,

50: user I / F module, 51: mosaic building module,

52: navigation module, 53: cuttrace module,

54: stage control module, 55: laser control module,

56: CCD camera control module, 57: system driver module,

58: image conversion module,

70: slide, 72: transparent glass,

74: tissue section, 76: transparent membrane,

78: slide zero, SA: scan area

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

1 is a schematic appearance configuration diagram of a laser microdissection system to which the present invention may be applied. As shown in FIG. 1, a general configuration of a laser microdissection system to which the present invention can be applied is largely installed in a microscope device, a place of a microscope device, and a charge coupled device (CCD) camera for capturing an enlarged image by the microscope device. And a stage device for microscopic movement of slides loaded with tissue sections mounted on a microscope device, a sample collection device for collecting finely excised cell samples, a laser generating device, a laser / stage controller, and a computer device for managing the entire system. Can be.

In the above-described configuration, the microscope device may comprise an alternative lens 2, an objective lens 4, a dimming filter 9, a condenser 8 and a halogen lamp 16, which configuration is a microscope device. Since it is well known in the art, a detailed description thereof will be omitted.

The stage apparatus again mounts a slide (not shown) to scan a tissue section secured to the slide in a predetermined path or a stage top plate 5 for horizontally moving the slide to ablate a cell sample and a stage top plate for optical focusing. It may consist of a stage lower plate 7 for vertically moving 5). Here, the stage upper plate 5 is moved by an x-axis motor and a y-axis motor (not shown), and the stage lower plate 7 is moved by a z-axis motor (not shown), with respect to each axis of the stage upper plate 5. The minimum movement amount can be 1 [μm] or less.

The laser generating apparatus again includes a laser generator 14 for generating a laser beam having a predetermined wavelength, a laser optical channel 3 through which the laser beam generated by the laser generator 14 passes, and a laser in the laser optical channel 3. The laser focus lens 11 and the laser focus lens 11 which move in the longitudinal direction in the laser reflector 13, the laser condenser lens group 12, the laser optical channel 3 to refract the beam to maintain the focus of the laser beam. It may comprise a reflector 10 for refracting the laser beam focused by the toward the objective lens (4).

The laser / stage controller 21 consists of hardware for driving a laser generating device and a stage device.

The sample collection device includes a sample holder 6 movably mounted to the stage lower plate 7 and an x-axis motor (not shown) for moving the sample holder 6 to the optical axis of the microscope device with the sample container installed. It can be made by the bar, it is preferable to install a plurality, for example four, a plurality of sample collection boxes to be collected by separating the various types of cell samples by type.

The computer device includes a central processing unit, a main body such as RAM and ROM, and a main body including auxiliary memory such as a hard disk, and a computer main body including software such as an operating system and image processing / management programs. ), A monitor 18 for displaying a real time image captured by the CCD camera 1 or a stored image read from a predetermined memory, a keyboard 19 connected to the computer main body 17 for inputting and selecting necessary items from a user; It may be made including a mouse 20. In the figure, reference numeral 15 denotes a cooling fan that cools the heat generated by the laser generator.

In the above-described configuration, the CCD camera 1 has, for example, a sensor specification of 11.1 [mm] * 7.9 [mm], a pixel count of 1666 * 1200, and each pixel specification of 7.4 [µm] * 7.4 [µm]. Could be In this case, for example, when using an objective lens 4 of 10 magnification and a camera eyepiece of 0.74 magnification, an area having a size of 1 [µm] * 1 [µm] is 7.4 in the CCD sensor of the CCD camera 1. [Μm] * 7.4 [μm], which will be 1 pixel on the monitor screen. In contrast, in the case of using a 4x objective lens 4 and a 0.74 magnification camera eyepiece, an area of 2.5 [μm] * 2.5 [μm] is 7.4 [μm] * 7.4 in the CCD sensor of the CCD camera 1 [Μm] and this will be 1 pixel on the monitor screen.

FIG. 2 is an electrical functional block diagram of a laser microsurgery system to which the present invention can be applied. For convenience, the same reference numerals are given to those of the mechanical configuration shown in FIG. 1. As shown in FIG. 2, the electrical functional blocks of the laser microdissection system to which the present invention can be applied include the computer hardware 30, the CCD camera 34 connected to the computer hardware 30, and the mouse 20 as a user input interface. ), A system control software (31) mounted on a computer, which controls the whole microablation system of the present invention as a whole, a system hardware control unit (32) for hardware control of a laser generating device, a stage device, and a sample collection device, and system hardware. A laser focus control unit 35, a laser power control unit 36, a laser beam switch 37, a stage driver 38, and a collection box driver 39 which perform bidirectional communication with the control unit 32 to perform a given function, respectively. Can be done.

Figure 3 is a functional block diagram showing the operation method of the laser microdissection system of the present invention in a module unit. As illustrated in FIG. 3, a method of operating the laser ablation system according to the present invention will be described in units of modules. The user interface module 50, the mosaic image construction module 51, the navigation module 52 for the overall image, The cuttrace module 53, the stage control module 54, the laser control module 55, the CCD camera control module 56, the system driver module 57, and the image conversion module 58 may be formed.

The user interface module 50 interfaces user input items such as the maximum resolution of the CCD camera 1, the magnification of the objective lens 4, the size of the cell tissue, the setting of the scanning area or the selection of the ablation area, etc., from the user.

The mosaic image construction module 51 combines the images of the local regions obtained by the CCD camera 1 to display a comprehensive image in the form of a mosaic. The overall image thus obtained is formed in a reduced image form and the entire image is reduced in a reduced scale. It is designed to be connected without breaking. The reduced accumulation is then automatically calculated so that the overall image is properly displayed on the monitor screen. Since the overall image file for searching is stored in the form of a bitmap image (bmp) file in the computer memory, it can be repeatedly read. That is, the user can open a search file and bring up a screen by using a mouse. At this time, a part of the screen can be selected for detailed observation. The selected part is displayed as a navigation picture in the view window square window, the size of which is automatically calculated according to the size of the computer monitor. The specific location of the view window, for example the upper left corner point, is the starting point for the navigation module 52 for the overall image.

The search module 52 for the overall 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 instructed by the mosaic image building module 51, which may be stored in the RAM of the computer main body on a 1: 1 scale, for example. In this state, when the user presses the zoom button, the user can enlarge the accumulation ratio up to 8: 1, and can also move the image in a desired direction by dragging the image through a mouse operation. At this time, adjacent video files (images next to the selected part) are automatically stored in the RAM of the computer main body during the movement and appear on the monitor screen with the currently selected accumulation. In this process, unnecessary video files are deleted from the RAM.

The cuttrace module 53 allows a user to select a required collection box by operating a mouse and to draw a laser cutting path required for an image displayed on a monitor screen. The cuttrace module 53 also automatically assigns a color for cutting path display according to the type of cell sample at the same time as the collection selection. There is a match between the cleavage path drawn in this way and the collection bin where the cleaved cell sample falls off. On the other hand, the cutting path is displayed along with the cell image according to the accumulation selected by the user on the monitor screen, the coordinates of the cutting path may be stored in a computer memory, for example, a hard disk.

Figure 4 is a schematic view showing a cross-sectional structure of the slide that can be applied to the operating method of the laser microdissection system of the present invention. As shown in FIG. 4, the slide 70 has a tissue section 74 placed on top of a transparent glass plate 72 having a predetermined size, and the tissue membrane 74 protects the tissue section 74. The slide 70 is mounted on the stage top plate 5 while being turned upside down in use.

FIG. 5 is a flowchart illustrating a process of acquiring a comprehensive image of a scan area from a slide in a method of operating the laser microdissection system of the present invention. As shown in Fig. 5, in step S10, the stage top plate 5 (hereinafter, simply referred to as "stage" since all stages shown in Figs. The optical axis is placed at a predetermined position of the stage, which position may be determined in relation to the zero point (slide zero point) to be displayed on the slide 70.

6 is a view for explaining a process of displaying the zero point on the slide in the operating method of the laser microdissection system of the present invention. As shown in FIG. 6, the stage 5 is formed with a fixing member 5a for fixing the slide 70 at a predetermined position so that the slide 70 can be placed at a substantially fixed position on the stage 5. do.

Of course, because of this structure, it is also conceivable to make a predetermined position of the stage 5, for example, a right angle intersection point of the fixing member 5a as shown in FIG. 6 as the zero point of the stage 5 and the zero point of the slide 70. Can be. However, in the case of cutting a cell sample at a resolution of about 1 [μm], fine foreign matter is interposed between the fixing member 5a and the slide 70 or between the fixing member 5a and the slide 70 due to vibration or the like. If there is a small gap in the two of these zero point is shifted, it is impossible to perform an accurate ablation on the desired area. For this reason, in the present invention, the physical zero is displayed on the slide 70 to be taken as the zero point of the absolute coordinate system in the ablation process. As a result, since the zero point does not change, today, the ablation area is obtained by obtaining a comprehensive image of the tissue section 70. Even if the ablation is performed on another day, accurate ablation can be performed for the desired area.

Next, in step S12, the physical zero is displayed on the slide 70 by irradiating a laser beam while moving the stage 5 as predetermined. In this step S12, the physical zero is orthogonal cross (+). It could be implemented as an intersection of markers. Moreover, such a zero point 78 is preferably formed in a portion of the slide 70 that is not occupied by the tissue section 74 in order to reduce errors in the image recognition process.

In one example, the zero point of the stage 5 is the orthogonal point of the fixing member 5a, and the x and y axes at the zero point of this stage 5 are irrespective of whether this point is the upper left corner point of the actual slide 70. By using the points on the slide 70 spaced 10 [mm] apart from each other, the cross mark (+) can be displayed. In this case, when the slide zero 78 is recognized in the ablation process to be described later, only the portions of 10 [mm] from the zero point of the stage 5 to the x-axis and the y-axis may be searched intensively. You will be able to recognize it.

Next, in step S14, a scanning area is input from a user while capturing a real-time image by a CCD camera and displaying the same on a monitor screen. The scanning area is, for example, a rectangular window set by a user by dragging a mouse. It can be determined by taking the upper left corner point and the lower right corner point. The dashed-dotted line in FIG. 6 illustrates the scan area SA set by the user.

Next, in step S16, the absolute coordinate value of the scan area SA is calculated and stored on the basis of the slide zero 78, and in step S18, the scan area SA is divided into local areas by a predetermined matrix. . The matrix may be automatically determined by the maximum resolution of the CCD camera, the magnification of the objective lens, the size of the tissue section, or the like.

Next, in step S20, the image of each local area is captured and stored in the computer memory while moving the stage. In step S22, the captured local unit image is collectively constructed and the mosaic image is stored in the computer memory.

On the other hand, unlike shown in Figure 5, it is also possible to produce a slide using a slide substrate in which the zero point of the absolute coordinate system is displayed in advance, in this case, a laser beam, which is a non-contact tool may be used as a zero point display tool, such as a diamond cutter Contact tools may be used, and in some cases it may be possible to mark them using dyes. As described above, the display position may be displayed at a predetermined position of the slide substrate, for example, 10 [mm] on the x-axis and 10 [mm] on the y-axis from the upper left corner point of the slide substrate. will be.

On the other hand, when using a slide substrate in which the zero point of the absolute coordinate system is displayed in advance, a process of recognizing the zero point displayed on the slide substrate before setting in step S14 of FIG. 5 and setting the zero point of the absolute coordinate system is required. The step S12 of the flowchart shown in FIG. 9 will be changed to the content of recognizing the zero point and setting the zero point of the absolute coordinate system.

FIG. 7 is a view for explaining a process of setting a scan area and a local area in an operating method of the laser microdissection system of the present invention, and illustrates that the scan area SA is composed of a local area divided into a total of 8 rows * 8 columns. Doing. The process of capturing a local image will be described in more detail. For example, after capturing an image of a local area in a first row and a first column, the image is captured after the upper left corner of the scan area SA is set as a zero of a relative coordinate system. For example, save the file as "file name 1111.JPG". Next, after capturing the image of the local area of the first row and the second column while moving the stage to a relative coordinate value corresponding to the first row and the second column, and storing it as a file named, for example, "file name 1211.JPG" This operation is repeated for the local area in the entire scan area SA. Finally, the overall image is saved as a file named "file name. BMP".

Although not shown, when the user selects an ablation area within the scan area SA, a relative coordinate value on the scan area SA with respect to the ablation area is obtained to determine the type of ablation area, that is, the ablation path display color. It will be saved as a separate coordinate file in computer memory. In this case, the zero point of the relative coordinate value may be, for example, the upper left corner point of the scan area SA.

8 is a flowchart illustrating a process of performing a fine ablation operation on a slide in the method of operating the laser microablation system of the present invention. As shown in FIG. 8, in step S30, a zero, that is, a cross mark (+), is recognized on a slide placed on a stage for an ablation operation using, for example, a correlation navigation method used in cruise missile technology. This is then set to the zero of the absolute coordinate system in the ablation process. However, the slide zero can be quickly and easily recognized using the method described in connection with the above-described step S12.

Next, in step S32, the overall image of the scan area pre-stored in the computer memory is read in response to the slide. In step S34, the absolute coordinate values of the scan area SA pre-stored in the computer memory are read. In step S36, the scan area is read. All ablation zones present in the (SA) are read from the computer memory and checked, and classified by color.

Next, in step S38, the relative coordinate values of the scan areas of all the ablation areas are read from the computer memory, and in step S40, the collection bin corresponding to the first kind is moved directly below the optical axis. Again, in step S42, the laser beam is irradiated while moving the stage to collect and collect the cell sample in the ablation zone, and then, in step S44, it is determined whether all ablation work for the homologous cell sample is completed.

As a result of the determination in step S44, when all the ablation work on the allogeneic cell sample is not completed, the process returns to step S42, and when it is completed, proceeds to step S46 to determine whether the ablation work on all kinds of cell samples is completed. do. As a result of the determination in step S46, when the ablation work for all kinds is not completed, the process returns to step S40, and when the ablation work for all kinds is completed, the ablation process is ended. As described above, in the method of operating the microdissection system of the present invention, the microdissection operation is performed without alternating with the heterologous species and allogenes, and the excision operation is performed by minimizing the movement of the collection container by performing the microdissection operation on all cell samples. In addition to increased accuracy, the time required for fine cutting can be shortened.

The operation method of the laser microdissection system of the present invention is not limited to the above-described embodiment, and can be modified in various ways within the scope of the technical idea of the present invention. For example, the slide that can be used in the present invention may not only have the above-described structure and material, but also may be variously modified within a range that does not interfere with the operation method of the present invention.

According to the operating method of the laser fine ablation system of the present invention as described above, ablation is performed by constructing an absolute coordinate system by the physical zero displayed on the slide, and performing an ablation site selection operation and an ablation operation based on the absolute coordinate system. There is an effect that can remove the time and space constraints between site selection and ablation. For example, according to the method of the present invention, after an experimenter sends a general image file scanned on a certain day to another scholar, the other day, the scholar receives a result file showing an ablation area on the overall image, and On other days, it is possible to perform an operation such as performing an ablation based on the result file.

Claims (7)

Microscope device, CCD camera installed in the microscope device to capture the magnified image by the microscope device, Stage device installed on the microscope device to move the slide to which the tissue sections are fixed, Sample collection device for collecting the finely excised cell sample In the operating method of the laser microdissection system comprising a laser device and a management computer for irradiating a laser beam toward the optical axis of the microscope device, Mounting the slide at a predetermined position on the stage device; Moving the stage device to recognize a zero point present on the slide and setting the zero point for the ablation operation; Reading coordinate images of tissue sections fixed to the slide and coordinate values based on the zero point of the ablation zone included in the aggregate images from a memory of a computer; and And cutting the cells by irradiating a laser beam while moving a stage device according to the coordinate value of the ablation area based on the zero point. The method of claim 1, wherein the obtaining of the comprehensive image comprises: Mounting the slide at a predetermined position on the stage device; Moving the stage apparatus such that the optical axis of the microscope apparatus is at a predetermined position of the stage apparatus; Displaying the zero point by irradiating a laser beam onto a slide while moving a stage device as predetermined; Receiving a scan area on a monitor screen of a computer; Calculating coordinate values of the scan area based on the zero point and storing the coordinates in a computer memory; Partitioning the scan area into a predetermined matrix; Moving the stage device and capturing an image of each local area partitioned by the matrix and storing the image in a memory of a computer; and A method of operating a laser microsurgery system comprising the steps of constructing and storing a collective image by collectively capturing captured local unit images. A slide according to claim 1, wherein the zero point slide is used, The acquisition process of the overall image, Mounting the slide at a predetermined position on the stage device; Moving the stage device to recognize the zero point and setting the zero point for the scan operation; Receiving a scan area on a monitor screen of a computer; Calculating coordinate values of the scan area based on the zero point and storing the coordinates in a computer memory; Partitioning the scan area into a predetermined matrix; Moving the stage device and capturing an image of each local area partitioned by the matrix and storing the image in a memory of a computer; and A method of operating a laser microsurgery system comprising the steps of constructing and storing a collective image by collectively capturing captured local unit images. 4. The method according to claim 2 or 3, wherein the zero point is displayed on a portion of the slide not occupied by the tissue section. The laser ablation according to claim 2 or 3, further comprising converting and storing the ablation area into a relative coordinate value with respect to the scan area when the ablation area is set in the overall image. How the system works. The sample collection device according to claim 5, wherein the sample collection device includes a sample collection container which is movable in the optical axis of the microscope device and is provided with a plurality of cell samples. The ablation zone is set to be divided according to the type of cell sample operating method of the laser microscopic ablation system, characterized in that the ablation work for the cell sample of the same type can be performed collectively. On the slide that is mounted on the movable stage and physically marked on the slide to which the tissue section is fixed, the image is recognized as the zero point of the coordinate system, and the coordinate value based on the zero point is set for the ablation area displayed on the image where the tissue section is captured. Operating method of the laser microdissection system to perform a fine ablation while moving the stage based on the coordinate value in the given state.