JPH10127267A - Micro-manipulator system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a micro-manipulator capable of accurately specifying cutting sites for cutting specific sites of a chromosome and facilitating the treatment. SOLUTION: This micro-manipulator detects specific cites of a chromosome, which are colored with a fluorescent dye, from image signals obtained through a fluorescent microscope and a TV camera and specifies cutting cites of the chromosome (step S5), detecting the fluorescent-colored tip of a cutting needle from image signals obtained through an imaging means (step S6), calculating the moving amounts of a cutting unit needed for cutting the specified sites of the chromosome by the cutting needle (step S7), cutting the first cutting site by controlling the manipulator which holds the cutting needle (step S9) and subsequently cutting the second cutting site (step 11).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is suitable for micromanipulator systems, in particular, for cutting a desired position of a chromosome in molecular biology such as genetic engineering, genetic diagnosis such as genetics, immunochemistry, and histochemistry. The present invention relates to a micromanipulator system.

[0002]

2. Description of the Related Art In a clinical site of genetic diagnosis, a specific site of a chromosome is cut to perform a predetermined treatment. For such an operation, a micromanipulator holding a cutting tool for cutting a chromosome at the tip is used. A general micromanipulator system includes a microscope for enlarging an image of a small sample such as a chromosome and observing the operation contents of such a micromanipulator. Also, a TV camera for photographing an image magnified by a microscope and a CRT for displaying the image
A display and the like are provided. In the field of view of the microscope, operate a micro instrument such as a micro needle, a capture needle, and a glass tube for cutting held by a plurality of micro manipulators,
The micro sample is displayed on an RT display or the like, and predetermined processing is performed while observing the displayed content. The micromanipulator has an instrument holder at its tip for holding a micro instrument, and has moving means for moving the instrument holder in a three-dimensional direction. The amount of movement of the moving means is determined, for example, by support from an operation unit having a joystick for X and Y axes that can be tilted in the front-back direction and a rotary dial for Z axis.

[0003]

The use of the micromanipulator system as described above to perform processing on a minute sample by manual operation requires skill. In particular, when a cutting tool such as a glass tube having a sharp tip is held by a micromanipulator to cut a specific portion of a chromosome, it is necessary to position the cutting tool at a predetermined position of the chromosome and reciprocate a plurality of times. Is complicated. Further, it is difficult for a skilled person to determine which portion of the chromosome is to be cut and at what width the cutting is to be performed, and at present it depends on the experience and intuition of the operator. Furthermore, moving the tip of the cutting tool held by the micromanipulator to a target position requires experience with this operation.

[0004] It is an object of the present invention to provide a micromanipulator that, when cutting a specific site of a chromosome, reliably identifies the site of the cut and facilitates processing.

[0005]

A micromanipulator system according to the present invention comprises: a fluorescence microscope;
The micromanipulator includes a micromanipulator, a cutting site detecting unit, a cutting tool detecting unit, and a control unit. Fluorescence microscopes magnify images of objects. The imaging means converts the image of the object enlarged by the fluorescence microscope into an image signal. The micromanipulator holds a cutting tool for cutting an object movably in a three-dimensional direction. The cleavage site detection means detects a specific site of the chromosome colored by a fluorescent dye from the image signal obtained by the imaging means, and specifies a chromosome cleavage position. The cutting tool detecting means detects a tip position of the cutting tool colored with a fluorescent dye from an image signal obtained by the imaging means. The control means moves the cutting tool necessary to cut a specific portion of the chromosome by the cutting tool based on the cutting position detected by the cutting site detecting means and the tip position of the cutting tool detected by the cutting tool detecting means. The amount is calculated and the movement of the micromanipulator is controlled.

In such a micromanipulator system, FISH (Fluorescence in Situ Hybridi
The specific position of the chromosome is fluorescently colored with a probe having a fluorescent dye using the zation method or the like, and the image of the chromosome is magnified by a fluorescent microscope. The image of the chromosome is converted by the imaging means into an image signal together with an image of the tip of the cutting tool held by the micromanipulator. The specific site of the chromosome is luminescent by the fluorescent dye of the probe,
By comparing the image signal with a predetermined threshold value, it becomes possible to identify a specific part to be cut. The tip position of the cutting tool is also colored by the fluorescent dye, and similarly, it becomes possible to identify the tip position of the cutting tool in the image signal. Based on the position of the specific portion of the chromosome identified in this way and the tip position of the cutting tool, the amount of movement of the cutting tool necessary for performing cutting is calculated, and the movement of the micromanipulator is controlled. Therefore, it is possible to reliably and accurately cut a desired position of a chromosome.

[0007]

FIG. 1 shows a micromanipulator system according to an embodiment of the present invention. In the figure, the micromanipulator system has micromanipulators 4 and 5 arranged on the sides of the microscope 3, and the microscopes 3 are connected via coupling frames 6 and 7, respectively.
It is fixed to. The microscope 3 is a so-called fluorescent microscope, and is capable of observing and discriminating a site emitting light with a fluorescent dye.

[0008] Although only one pair of micromanipulators is shown, one or more pairs of micromanipulators can be arranged behind micromanipulators 4 and 5 (vertically below the sheet). The micromanipulators 4 and 5 are controlled according to the operation of joysticks 10 and 11 provided in the operation units 8 and 9, respectively. The operation units are provided corresponding to the micromanipulators, and when four micromanipulators are arranged, four operation units are provided.
9 is shown.

The microscope 3 has an operation table 12 at the center thereof, and the operation table 12 has a slide glass 13 on which a small sample such as a cell chromosome is placed. Operation console 1
An objective lens (not shown) is arranged below the camera 2 and a TV camera 14 is connected to the other end of the objective lens. The operation console 12 can be driven in a horizontal direction (X and Y axis directions) and a vertical direction (Z axis direction) by a drive mechanism (not shown). Further, the microscope 3 is provided with an eyepiece 15 so that an image obtained by the objective lens can be confirmed.

The micromanipulator 4 is provided with an arm 17 having an instrument holder 16 at its tip, protruding from the operation table 12. The tool holder 16 holds a cutting tool 18 made of a cutting glass tube or the like whose tip is sharply formed. The instrument holder 16 is connected to an injector or the like (not shown). In addition, in order to move the cutting tool 18 held by the tool holder 16 to the micromanipulator 4,
A drive mechanism is provided in the horizontal direction (X and Y axis directions) and the vertical direction (Z axis direction). The driving mechanism includes an X-axis stepping motor 19 driven in the X-axis direction, a Y-axis stepping motor (not shown) driven in the Y-axis direction, a Z-axis stepping motor 20 driven in the Z-axis direction, and X, Y, Z
It is composed of guide rails (not shown) for guiding in each axial direction.

An arm 22 having an instrument holder 21 at the tip is similarly provided on the micromanipulator 5.
It protrudes from two sides. The instrument holder 21 holds a small instrument 23 such as a cutting glass tube, an injection pipette, or a suction pipette similar to the cutting tool 18.
The instrument holder 21 is connected to an injector (not shown) or the like. In addition, the micromanipulator 5 is used to move the small instrument 23 held by the instrument holder 21.
A drive mechanism is provided in the horizontal direction (X and Y axis directions) and the vertical direction (Z axis direction). This driving mechanism includes an X-axis stepping motor 24 driven in the X-axis direction, a Y-axis stepping motor (not shown) driven in the Y-axis direction, a Z-axis stepping motor 25 driven in the Z-axis direction, and X, Y, Z
It is composed of guide rails (not shown) for guiding in each axial direction.

The joystick 1 of the operation units 8 and 9
Numerals 0 and 11 can tilt in the front-rear and left-right directions with respect to the vertical axis direction (vertical direction in FIG. 1), and support the control amounts of the micromanipulators 4 and 5 in the horizontal direction (X and Y axis directions). Can be. A rotary dial is provided on the upper part of the joysticks 10 and 11, and by operating the rotary dial, it is possible to give a control amount in the vertical direction (Z-axis direction) of the micromanipulators 4 and 5. . The operation units 8 and 9 are provided with key switches for performing other controls.

Operation signals from the operation units 8 and 9 are input to the control unit 26. The control unit 26 outputs a control signal for driving the micromanipulators 4 and 5 according to the operation signals of the operation units 8 and 9. Further, the control unit 26 has a TV set.
The image of the camera 14 is input, and this image is displayed on the monitor 27.

As shown in FIG. 2, the control unit 26 is provided with a control unit 28 composed of a microcomputer including a CPU, a RAM, a ROM and the like. The control unit 28 includes the TV camera 14, the control unit 8
(9) driving units 29 and 30 for driving the respective motors of the micromanipulators 4 and 5 in three axial directions, a memory 31 for storing various set values and the like, a video RA
An adder 33 for superimposing the output of the M32 and the video RAM 32 and the image captured by the TV camera 14
Is connected. The video RAM 32 stores information such as a display position of a reference point for focusing the cutting tool 18 and the micro tool 23 on the image, information for displaying a scale corresponding to the magnification, and the like corresponding to the display position. The monitor 27 that displays the superimposed image is connected to the adder 33.

Next, the operation of this embodiment will be described with reference to the flowchart shown in FIG. After the power is turned on, initialization is performed in step S1. Here, for example, the drive units 29 and 30 of the micromanipulators 4 and 5 are set to the initial positions. In step S2, it is determined whether or not the mode is the automatic cutting mode. This automatic disconnection mode can be set by a key switch provided on the operation unit 8 or 9, and can be configured to be set according to a menu screen displayed on the monitor 27. .

If it is determined in step S2 that the automatic cutting mode has not been set, the process proceeds to step S3. In step S3, the micromanipulators 4 and 5 are controlled based on the operation of the operation units 8 and 9.
In step S4, other processing is performed. If it is determined in step S2 that the automatic disconnection mode has been set, the process proceeds to step S5. In step S5, the microscope 3 and the TV camera 1 set to the mode for detecting the fluorescent dye
The position of the chromosome break is recognized from the image signal obtained through Step 4. Here, the micro sample placed on the slide glass 13 of the microscope 3 is a chromosome whose specific site is colored by a fluorescent dye. A method of coloring a chromosome is to dye a probe adsorbed at a specific site with a fluorescent dye and react the probe with a specific site on the chromosome, so-called FISH (Fl).
uorescent in Situ Hybridization) method.

An image signal input to the control unit 28 via the TV camera 14 is displayed on the monitor 27 as, for example, an image frame 41 as shown in FIG. In the image frame 41 of FIG.
A colored portion 43 colored with a fluorescent dye by the fluorescent probe is displayed. The horizontal direction of the image frame 41 is X
The brightness signal level of each pixel is compared with a threshold level Th, and the coordinates of a pixel having a brightness signal level larger than the threshold level Th are obtained. In FIG. 4, coordinates x1, x2,
The cutting position is recognized by obtaining y1 and y2.

In step S6, the position of the tip of the cutting tool 18 held by the tool holder 16 of the micromanipulator 4 is recognized. As shown in FIG. 5, a cutting needle 44 formed of a glass tube having an internal hollow 45 is provided at the tip of the cutting tool 18, and the cutting needle 44 has a structure having a sharp tip of 1 μm or less. I have. In addition, this cutting needle 4
A liquid having a fluorescent dye is sucked into an inner hollow 45 of the glass tube constituting the container 4 by an injector or the like.

The cutting needle 44 is input to the control unit 28 as an image signal by adjusting the focus of the microscope 3, and
The two images are superimposed to form one image frame 46. Here, the internal hollow 4 of the cutting needle 44
5, a fluorescent dye is inhaled, and the coordinates of the tip position of the cutting needle 44 can be recognized in the same manner as in the case of recognizing the colored portion 43 of the chromosome 42.

In step S7, the micro-tool holding the cutting tool 18 is determined based on the coordinates of the coloring portion 43 of the chromosome 42 recognized in step S5 and the coordinates of the tip position of the cutting needle 44 recognized in step S6. The control amount of the manipulator 4 is calculated. In step S8, a setting input of how many times the reciprocating movement of the cutting needle 44 is performed when the cutting is performed is waited and stored. When there is no setting input of the number of reciprocating movements, a default value is set to 2-3 times as the set number of times.

In step S9, a first cutting operation is performed.
In the first cutting operation, the cutting needle 44 is moved to the first cutting position 51 shown in FIG. 6 based on the control amount calculated in step S7.
Is moved, and the micromanipulator 4 is controlled such that the cutting needle 44 reciprocates a set number of times in the direction of the arrow.
Here, the position of (x1, y1)-(x2, y1) in FIG. 4 corresponds to the first cutting position 51, and the tip of the cutting needle 44 is controlled to reciprocate at this position.

In step S10, it is determined whether the first cutting operation has been completed. If the first cutting position is not completely cut in the first cutting operation in step S9, the first cutting operation is performed.
It is determined that the cutting operation has not been completed, and the process proceeds to step S8. If it is determined that the first cutting operation has been completed, the process proceeds to step S11. In step S11, a second cutting operation is performed. In the second cutting operation, the cutting needle 44 is moved to the second cutting position 52 shown in FIG. 6 based on the control amount calculated in step S7, and the cutting needle 44 similarly reciprocates a predetermined number of times. The micromanipulator 4 is controlled. Here, similarly, (x1, y2)-(x
The position of (2, y2) corresponds to the second cutting position 52, and the tip of the cutting needle 44 is controlled to reciprocate at this position.

In step S12, it is determined whether the second cutting operation has been completed. If the second cutting position is not completely cut in the second cutting operation in step S11, it is determined that the second cutting operation is not completed, and step S13 is performed.
Move to In step S13, a setting input of the number of reciprocations for continuing the second cutting operation is waited and stored as the set number. At this time, if there is no setting input, the default value is set as the set number. Thereafter, the process proceeds to step S11 to continue the second cutting operation.

If it is determined in step S12 that the second cutting operation has been completed, the process proceeds to step S4. At this time, as shown in FIG. 7, the chromosome 42 is such that the colored portion 43, which is a specific site for cutting,
The cutting is performed at the second cutting position 52. Each of the cutting positions 51 and 52 emits light with a probe having a fluorescent dye to specify the position, so that the desired position of the chromosome can be reliably cut, and there is no variation in operation by the operator.

[Other Embodiments] (A) In the above embodiment, the chromosome 42 is placed so that its length direction is parallel to the Y-axis direction on the image frame, and the cutting needle 44 is aligned with the X-axis. Although it is cut by reciprocating in parallel, the chromosome 42 can be arranged in any direction. In this case, the coordinates of the position where the cutting is performed are represented by (x1, y
1), (x2, y2), (x3, y3), (x4, y
4), it is necessary to control the drive unit 29 of the micromanipulator 4 holding the cutting tool 18 as a combined movement in the X-axis and Y-axis directions. (B) The cutting needle 44 may have a configuration in which a fluorescent dye is applied to the surface of the glass tube to emit light on the surface. (C) Since this operation is performed by recognizing the coordinates, it is possible to perform this operation in a bright field, and in this case, it is easy to visually confirm the state of the operation.

[0026]

According to the present invention, it is possible to accurately and accurately recognize a chromosome break and perform a cutting operation automatically and reliably, without having to rely on the experience and intuition of a skilled person.

[Brief description of the drawings]

FIG. 1 is a front view of a micromanipulator system to which an embodiment of the present invention is applied.

FIG. 2 is a block diagram of a control unit.

FIG. 3 is a control flowchart of a control unit.

FIG. 4 is an explanatory diagram of a monitor image.

FIG. 5 is an explanatory diagram of a monitor image.

FIG. 6 is an explanatory diagram of a monitor image.

FIG. 7 is an explanatory diagram of a monitor image.

[Explanation of symbols]

 Reference Signs List 3 microscope 4 micromanipulator 8 operation unit 10 joystick 12 operation table 13 slide glass 14 TV camera 15 eyepiece 16 tool holder 18 cutting tool 26 control unit 27 monitor 28 control unit 29 drive unit 41 image frame 42 chromosome 43 coloring part 44 cutting needle 45 Internal hollow 51 First cutting position 52 Second cutting position

Claims (1)

[Claims] 1. A fluorescence microscope for enlarging an image of an object, an imaging means for converting an image of the object enlarged by the fluorescence microscope into an image signal, and a cutting tool for cutting the object is three-dimensional. A micromanipulator that movably holds in a direction, a cleavage site detection unit that detects a specific site of a chromosome colored by a fluorescent dye from an image signal obtained by the imaging unit and identifies a cleavage position of the chromosome, A cutting tool detecting means for detecting a tip position colored by a fluorescent dye of the cutting tool from an image signal obtained by the imaging means; a cutting position detected by the cutting site detecting means; and a cutting tool detecting means detecting the cutting position. Based on the cutting tool tip position,
Control means for calculating the amount of movement of the cutting tool necessary for cutting the specific portion of the chromosome with the cutting tool and controlling the movement of the micromanipulator.