WO2005096063A1

WO2005096063A1 - Manipulator device, manipulator, and method of operating treatment tool using manipulator device

Info

Publication number: WO2005096063A1
Application number: PCT/JP2005/006079
Authority: WO
Inventors: Nobuhiro Kita; Hideya Kitagawa; Akira Yagi; Yasushi Aono
Original assignee: Olympus Corporation
Priority date: 2004-03-30
Filing date: 2005-03-30
Publication date: 2005-10-13
Also published as: JP4331748B2; JPWO2005096063A1

Abstract

A manipulator device, a manipulator, and a method of operating a treatment tool using the manipulator device. The manipulator device operating an operation object under observation by an observational optical system comprises the treatment tool operating the operation object, a tip part involved in the treatment tool and coming into contact with or moving closest to the operation object, and a detection means detecting the change of light including scattered light from the tip part illumination by an illuminating light which occurs by the displacement of the tip part.

Description

MANIPULATOR DEVICE, MANIPULATOR, AND METHOD OF OPERATING TREATMENT TOOL USING MANIPULATOR DEVICE

Technical field

The present invention relates to a manipulator device, a manipulator, and a method for operating a treatment tool using the manipulator device.

This application claims the priority of Japanese Patent Application No. 2004-100820 filed on Mar. 30, 2004, the contents of which are incorporated herein by reference.

Background art

[0002] Conventionally, as a means for introducing a gene into a biological sample such as a cell as an object and expressing the gene, a physical method such as a microinjection method, an electoru-portion method, and a particle gun method has been used. And a method using a cell endocytosis method such as a calcium phosphate method or a ribofusion method are known.

Among these methods, in the microinjection method as described in Patent Document 1 below, a DNA solution is directly injected into individual cells, so that a gene can be selectively introduced into any cell. it can. In this method, if the tip of the treatment tool of the manipulator device is thick, the damage to the biological sample is increased, and the survival rate after the operation is not good, which may hinder the experiment after introduction.

Therefore, for example, in Patent Document 2 below, as a treatment tool, no damage is caused when inserted into a cell! / An ultrafine needle is used, and a gene or a substance involved in gene expression is transferred to the needle. In this state, the gene is introduced into the cell by inserting and retaining the cell in that state.

Patent Document 1: JP-A-06-109979

Patent Document 2: JP-A-2003-325161

Disclosure of the invention

Problems to be solved by the invention

[0003] Generally, a manipulator device for performing gene transfer as described above is a target. Selection of cells is performed under a light microscope. Similarly, the tip of the treatment tool for treating cells is also detected by the optical microscope, and the relative positional relationship (in-plane position and height) with the target cells is controlled.

Here, as described in Patent Document 2 described above, if the diameter of the distal end of the treatment tool is reduced to reduce damage to cells, it becomes difficult to detect the distal end of the treatment tool with an optical microscope. Become. In Patent Document 2, an atomic force microscope (AFM) is added to measure the distance between the treatment tool and the cell by measuring the atomic force acting between the treatment tool cantilever and the cell. Is going. Therefore, there is a disadvantage that an expensive interatomic microscope, which is expensive and complicated in structure and operation, is required.

In addition, even with such a configuration, it is not possible to accurately align the cells with the distal end of the treatment tool.

[0004] The present invention has been made in view of the above problems, and has a low-cost and simple configuration, accurately obtains the position of the distal end of a treatment instrument of a mul- tiperator, and favorably controls the position of the distal end. The purpose is to carry out.

Means for solving the problem

[0005] The present invention relates to a mapper device for performing an operation on an operation target under observation by an observation optical system: a treatment tool for performing an operation on the operation target; Included in

A tip that comes into contact with or comes closest to the operation target; and a detecting unit that detects a change in light that is generated by displacement of the tip and that includes scattered light illuminated by illumination light. And a manipulator device comprising:

[0006] According to the manipulator device described above, even if the treatment tool is extremely fine and is difficult to observe, only the scattered light at the distal end of the treatment tool can be detected and used. The position of the distal end can be accurately obtained without the risk of damaging the treatment tool without the tool touching the object.

[0007] The manipulator device may further include an illumination optical system that selectively irradiates the illumination light to an area where the tip may exist, of the area observed by the observation optical system. preferable.

[0008] According to the manipulator device described above, the treatment tool tip is located in a predetermined area where positioning is desired. Only when the end reaches, the detecting means can detect a change in the image of the scattered light, so that the accuracy of detecting the position of the distal end of the treatment instrument can be improved.

[0009] In the above-mentioned manipulator device, it is preferable that the illuminating light is applied to an area where the tip portion may exist through the observation optical system.

[0010] In the above manipulator device, it is preferable that the illumination optical system generates evanescent light on a surface holding the operation target.

[0011] According to the manipulator device described above, the distal end of the treatment instrument can be brought close to a position as close as possible to 50 to 150 nm with respect to the surface holding the operation target. Since only the distal end of the treatment instrument is detected, the accuracy of detecting the position of the distal end of the treatment instrument can be significantly improved.

[0012] The manipulator device further includes first alignment means for aligning a position where the distal end can exist with an optical axis of the observation optical system, and the illumination optical system includes a dark-field illumination optical system. It is preferable that.

[0013] According to the manipulator device described above, the distal end of the treatment tool is moved using the dark-field observation optical system, so that the position far from the objective lens can be detected without directly detecting the illumination light from the illumination optical system. With the treatment tool described in (1), the image of the scattered light from the tip can be selectively detected with a simple microscope configuration.

[0014] The manipulator device further includes a second alignment unit that adjusts a position where the distal end portion may exist and a focal position of the observation optical system, and the detection unit is configured to irradiate the illumination light. It is preferable to detect a change in light including the scattered light passing through an opening provided at a position optically conjugate with the region.

According to the above manipulator device, by moving the distal end portion of the treatment tool using the confocal optical system, of the portion illuminated by the illumination optical system, it is optically conjugate with the opening. Only when the distal end of the treatment instrument reaches the position, the image of the scattered light can be formed on the detection means, so that the scattered light with the force of the distal end of the treatment instrument can be selectively captured, and , The accuracy of detecting the position of the tip can be greatly improved.

[0016] In the above-mentioned manipulator device, the observation optical system includes a disk having a plurality of the openings, and rotates the disk to rotate the disk through the openings. Preferably, an area where an end portion may exist is scanned and illuminated, and the detecting means detects a change in light including the scattered light passing through the opening provided in the disk.

According to the above manipulator device, by rotating the disk, the illumination light is scanned within the observation field of view, and the scattering from the distal end of the treatment tool reaching the focused portion of the observation optical system. Light can be reliably detected by the detecting means.

[0018] In the above manipulator device, the illumination optical system emits laser light and scans the laser light in a state where the laser light is converged in a region where the distal end portion may exist. It is preferable to detect a change in light including the scattered light that has passed through the opening provided at a position optically conjugate to the region where the laser light is converged.

[0019] According to the manipulator device described above, by scanning the observation light with the laser light, the scattered light from the distal end of the treatment tool that has reached a position optically conjugate with the opening is detected by the detection means. It can be detected reliably.

[0020] In the above-described manipulator device, the illumination optical system may be configured to irradiate at least a first area with a first illumination mode in which the illumination light is irradiated with the illumination light and a second area different from the first area with the illumination. It is preferable to switch between the second illumination mode for irradiating light.

According to the manipulator device described above, coarse and fine position detection of the distal end portion of the treatment tool, illumination of the distal end portion of the treatment tool, illumination of other portions, and the like are switched, respectively. Operations can be performed.

[0022] In the above-mentioned manipulator device, the illumination optical system irradiates the illumination light to a region where the distal end portion may exist in the first illumination mode, and the operation object in the second illumination mode. It is preferable that the object is irradiated with the illumination light.

According to the manipulator device described above, it is possible to detect an accurate position between the distal end portion of the treatment tool and the operation target.

[0024] In the above manipulator device, it is preferable that the distal end portion is smaller than the optical resolution of the observation optical system.

[0025] According to the manipulator device described above, if an extremely fine treatment tool is used, damage to the operation target can be suppressed to a minimum, and even with such a treatment tool, a method for detecting scattered light. Then, the position of the tip can be reliably detected. [0026] Preferably, the manipulator device further includes a position storage unit that stores a position of the distal end detected by the detection unit.

According to the manipulator device described above, if the position of the distal end of the treatment instrument is stored as, for example, a reference position, the position of the distal end of the treatment instrument can be reliably controlled by detecting the position once.

[0028] The manipulator device further includes a driving unit that adjusts a relative distance between the surface on which the operation target is held and the treatment tool, and the driving unit determines a result detected by the detection unit. It is preferable to control the movement of the treatment tool based on the above.

[0029] According to the manipulator device described above, the position of the distal end portion of the treatment tool detected by the detection means is used as a reference position, and the position adjustment without damaging the treatment tool and the operation on the operation target are performed. It can be carried out.

[0030] In the above manipulator device, the operation with the treatment tool may include inserting the treatment tool into the operation target, injecting a substance into the operation target, cutting the operation target, and cutting the operation target. It is preferable to include holding the object and physically or electrically stimulating the operation target.

In the above manipulator device, “operation” refers to various manipulations, including both cases where the distal end portion of the treatment tool touches and does not touch the target.

[0032] In the above manipulator device, it is preferable that the operation target includes a biological sample. Preferably, the biological sample is a living cell.

[0033] The present invention provides a manipulator used for the above manipulator device.

[0034] The present invention is a method for operating a treatment tool using a manipulator device, which performs an operation on an operation target under observation by an observation optical system: performing an operation on the operation target Illuminating a tip included in the treatment tool and in contact with or closest to the operation target with illumination light; and the tip illuminated by the illumination light, which is generated with displacement of the tip. Detecting a change in light including scattered light of force; and a method of operating a treatment tool using a manipulator device.

[0035] Illumination of a minute area using evanescent light, dark field illumination optical system, confocal optical system Although technologies such as these have existed in the past, there was no technical idea in the past to detect extremely fine needle tips using these technologies. According to the manipulator device described above, even if it is a fine treatment tool that is difficult to observe, the treatment tool touches an object only by detecting scattered light from the distal end of the treatment tool and using it. The position of the tip can be accurately determined without damaging the treatment tool.

[0036] The illumination optical system that selectively irradiates a predetermined area that can be observed by the observation optical system of the present invention with illumination light includes a surface that holds an operation target and is necessary for observation. An all-reflection illumination optical system that totally reflects light at the interface between the substrate that transmits light and the medium of the operation target can be applied.

According to this total reflection illumination optical system, the illumination light is totally reflected at the interface between the substrate that transmits light necessary for observation and the medium of the operation target. At this time, an evanescent wave traveling in the optical axis direction is generated in the medium of the operation target. Since this evanescent wave exists only in the region of about 50 to 150 nm from the interface between the substrate and the medium, when the treatment tool is lowered and the tip enters this region, the evanescent wave reflected at the tip becomes the observation optics. An image is formed on the detector via the system and detected. The region where the evanescent wave exists, that is, the region illuminated by the total internal reflection illumination optical system, is a region of about 50 to 150 nm.Since the region other than the distal end of the treatment tool is not illuminated, it is possible to detect only the distal end with good contrast. it can.

[0038] As the illumination optical system, a dark-field illumination optical system can be applied. In a dark-field illumination optical system, the illumination light is illuminated at an angle larger than the numerical aperture of the objective lens, and the light reflected on the horizontal plane does not directly enter the objective lens. However, if a minute object such as the distal end of the treatment tool exists in the illumination area, a part of the reflected light is incident on the objective lens, so that the distal end of the treatment tool can be detected.

As the illumination optical system and the observation optical system, a confocal optical system that illuminates one point of the sample and detects the reflected light intensity at only one point at the image forming position can be applied. When these are used, only the region focused on the sample is illuminated and an image is formed, so that the distal end of the treatment tool can be detected only when the distal end of the treatment tool is located in this region. As the confocal optical system, any of a disk scanning type and a laser scanning type can be applied. [0040] Further, the manipulator device of the present invention includes a driving means for adjusting a relative distance between the treatment object and the operation object or a surface on which the operation object is held, and the driving means is provided with the observation means. The relative distance between the surface on which the operation target is held and the treatment tool is adjusted based on the image of the treatment tool detected by the detection means selectively detecting through the optical system. .

[0041] In such a configuration, a mode for illuminating the surface holding the operation target and a mode for illuminating the treatment tool can be switched. Then, the operation object or the surface on which the operation object is held and the distal end of the treatment tool can be easily detected by illuminating them with the optimal illumination method. This makes it possible to adjust the relative distance between the treatment tool and the operation target.

The invention's effect

According to the present invention, it is possible to satisfactorily detect an image of the distal end portion of the treatment tool. For this reason, even if the treatment tool is so fine that it cannot be captured by a normal optical microscope, the predetermined operation can be performed on the operation target without causing the tip of the treatment tool to be in contact with the substrate and causing damage. It can be done safely. Therefore, the depth of insertion into the operation target can be accurately managed, and the operation on the operation target can be performed very efficiently.

Brief Description of Drawings

FIG. 1 is a diagram showing a first embodiment of a manipulator device of the present invention, and is a diagram showing a schematic configuration of the manipulator device.

FIG. 2 is a side sectional view schematically showing a total reflection illumination optical system of an inverted microscope.

FIG. 3 is a cross-sectional view illustrating a configuration of a cleaning unit.

FIG. 4 is a side view showing a configuration of a solution stage.

FIG. 5 is a flowchart showing a work procedure of the manipulator device according to the embodiment of the present invention.

FIG. 6 is a cross-sectional view showing a relationship between a biological sample and a needle in a total internal reflection state in an inverted microscope.

FIG. 7 is a view showing a second embodiment of the manipulator device of the present invention, and is a side sectional view schematically showing a dark-field illumination optical system of an inverted microscope provided in the manipulator device. FIG. 8 is a view showing a third embodiment of the manipulator device of the present invention, and is a side sectional view schematically showing a confocal optical system of an inverted microscope provided in the manipulator device.

FIG. 9 is a schematic diagram of a two-way disc used for a confocal optical system.

FIG. 10 is a view showing a modification of the third embodiment, and is a schematic view of a laser-scanning confocal optical system in an upright microscope.

Explanation of symbols

[0044] 1, la, lb, lc: manipulator device, 2: inverted microscope, 2a: total reflection illumination optical system, 3: XY stage, 3a: driving means, 4: sample container, 5: manipulator body, 6: main Mount, 7: Objective lens, 8: Eyepiece, 9: Light source, 10: Imaging means, 11: Image processing means, 12: Control unit, 13: Holding device, 14: Needle, 14a: Tip, 14b: Base, 14c: Power cord, 15: Positioning means, 16: Cleaning unit, 17: Solution table, 17a: Introduced substance solution, 21: Evacuation stage, 21a: Holding member, 22: Z stage, 24, 25: Driving means, 26: Supporting member, 29: fluorescent lamp, 30: wedge prism, 31: eccentric slit, 31a: adjusting means, 32: excitation filter, 33: mirror, 34: lens, 35: dichroic mirror, 41: cover, 41a: top surface Part, 42: Opening, 43: Ultrasonic cleaning tank, 44: Ultrasonic transducer, 45: Wiring code, 46: Electrode, 47: Wiring code, 50: Lamp, 51: Perforated mirror, 52: Field objective lens, 52a: Illumination light guide, 53: Imaging lens, 54: Beam splitter, 55: Po disk, 56: Rotary motor, 60: Laser light source, 61: Beam splitter, 62: Two-dimensional light Scanning mechanism, 63: pupil projection lens, 64: condenser lens, 65: confocal pinhole, 66: photodetector, 67: drive means (first alignment means), 68: drive means (second position) Matching means)

BEST MODE FOR CARRYING OUT THE INVENTION

The first embodiment according to the present invention will be described in detail with reference to the drawings.

FIG. 1 shows a configuration example of a manipulator device according to the present invention. This manipulator device 1 introduces an appropriate gene (introduced substance) into the nucleus of a cell (biological sample) C as an operation target existing in a medium Ca, and expresses a protein encoded by the gene. (See Figure 6).

The manipulator device 1 includes an inverted microscope (observation optical system) 2 and a manipulator body 5. It is the main component. The inverted microscope 2 is used for observing cells C and the like in an enlarged manner. The manipulator body 5 introduces the substance to be introduced into the cell C held via the medium Ca in the sample container 4 placed on the XY stage 3 of the inverted microscope 2 using a treatment tool such as a needle 14, for example. . Here, the sample container 4 is made of a translucent material or a transparent material, for example, made of glass.

[0047] The inverted microscope 2 includes an XY stage 3 on which a sample container 4 and the like are installed and which can be moved in two horizontal axes (front and rear and left and right), a main frame 6 supporting the XY stage 3, a sample container 4 The objective lens 7 (see Fig. 2) for observing the biological sample in the downward force, the eyepiece 8 for observing the image observed by the objective lens 7, and the light source 9 provided on the top of the main frame 6 Have. The inverted microscope 2 is provided with a lens for capturing an image of the biological sample observed by the objective lens 7 and an imaging unit 10 including a CCD camera. The imaging unit 10 is connected to a control unit 12 via an image processing unit (detection unit) 11. The XY stage 3 is provided with driving means 3a connected to the control unit 12. When the driving unit 3a is driven according to a signal from the control unit 12, the XY stage 3 moves in the front-back or left-right direction.

[0048] The manipulator main body 5 includes a holding device 13 for holding a substance to be introduced into cells C or the like of a biological sample. The holding device 13 cleans the needle (treatment instrument) 14 as an introduction means inserted into the cell C of the biological sample, the positioning means 15 for controlling the position of the needle 14, and the tip 14 a of the needle 14. A cleaning unit 16 for activation and a solution table 17 for accommodating a substance to be absorbed and held by the needle 14 are provided.

[0049] Needle 14 has a sharp distal end 14a and a base 14b having a larger diameter than distal end 14a. The tip 14 a of the needle 14 is smaller than the optical resolution of the inverted microscope 2. The needle 14 has its base 14b held by the positioning means 15. In this embodiment, at least the surface of the needle 14 is made of a conductive material such as platinum, and is connected to a power supply (not shown) via a power cord 14c. The positioning means 15 includes an evacuation stage 21 for holding the base 14b of the needle 14, a Z stage 22 for supporting the evacuation stage 21 movably in the Z direction (up and down directions), and the XY stage 3 described above. . Note that the XY stage 3 is also used as the inverted microscope 2. The evacuation stage 21 has a holding member 2 la that holds the base 14 b of the needle 14. The holding member 21a is connected to the driving means 24. When the driving unit 24 is driven according to a signal from the control unit 12, the holding member 21a moves in the horizontal direction. Driving means 25 is provided on the Z stage 22. When the driving unit 25 is driven according to a signal from the control unit 12, the Z stage 22 moves up and down. In other words, the Z stage 22 and the driving unit 25 constitute an elevating unit that moves the needle 14 up and down via the holding unit 21a and the evacuation stage 21.

The Z stage 22 is attached to the main gantry 6 via a support member 26.

As shown in FIG. 2, the total reflection illumination optical system 2a of the inverted microscope 2 is obtained by adding a wedge prism 30 and an eccentric slit 31 to an epi-illumination system for performing fluorescence observation. The total internal reflection illumination optical system 2a selectively irradiates illumination light to an area where the tip 14a of the needle 14 can be present among the areas observed by the inverted microscope 2, and a sample container holding cells C. Evanescent light is generated on the bottom surface of 4. In the total reflection illumination optical system 2a, the light emitted from the fluorescent lamp 29 passes through the excitation filter 32, the wedge prism 30, and the eccentric slit 31 in this order. Then, the light is reflected by the mirror 33, passes through the lens 34, is further reflected by the dichroic mirror 35, passes through the dedicated objective lens 7, and is incident on the back side (lower side) of the sample container 4.

[0052] Here, the position of the eccentric slit 31 can be changed by the adjusting means 31a. By changing the position of the eccentric slit 31, it is possible to freely set the incident angle of the illuminating light to the objective lens 7 at least in the vicinity of the critical angle, and eventually the incident angle to the back surface of the sample container 4. The illumination optical system of the present embodiment can perform illumination without depending on the spectral characteristics of the excitation filter 32 and the dichroic mirror 35.

As shown in FIG. 3, the cleaning unit 16 has a cover 41 for dust prevention. The cover 41 has a substantially square shape and a space inside. An opening 42 for inserting the needle 14 is provided substantially at the center of the upper surface 41a of the cover 41. The diameter of the opening 42 is a minimum size that allows a required amount of the needle 14 to be inserted in order to prevent dust. Inside the cover 41, an ultrasonic cleaning tank 43 and an ultrasonic vibrator 44 are arranged. The ultrasonic cleaning tank 43 holds a medium (for example, pure water, a neutral detergent, or an alkaline solution) enough to immerse the tip 14a of the needle 14 sufficiently. Ultrasonic vibrator 44 is ultrasonically cleaned It is attached to the lower part of tank 43. A wiring cord 45 is connected to the ultrasonic vibrator 44, and the wiring cord 45 passes through the cover 41 and is connected to a power supply (not shown).

When a detergent or a chemical is used as a cleaning liquid, neutralization and rinsing are sufficiently performed using a plurality of ultrasonic cleaning tanks 43.

As shown in FIG. 4, the solution stage 17 is provided with a plurality of recesses at predetermined intervals. For each of the plurality of recesses, for example, a plurality of different types of DNA (deoxyribonucleic acid)

) An introduction substance solution 17a such as a solution is dispensed. An electrode 46 is embedded in the solution table 17. The electrode 46 is connected to a power source (not shown) via a wiring cord 47. Instead of providing a concave portion for dispensing the solution 17a in the solution stage 17, the upper surface of the solution stage 17 is water-repellent and a plurality of hydrophilic regions are formed, and the solution 17a is held in the hydrophilic region. Anyway.

[0055] A procedure for causing the introduced substance to be held at the distal end portion 14a of the needle 14 and introducing the introduced substance into the biological sample using the manipulator device 1 will be described with reference to the flowchart shown in FIG.

First, the positioning means 15 shown in FIG. 1 is moved, and the needle 14 is inserted into the opening 42 of the cleaning unit 16, and as shown by the phantom line in FIG. Immerse in the medium in 43. In this state, power is supplied from the power supply to the ultrasonic vibrator 44 via the drive wiring 45, and the ultrasonic vibrator 44 is vibrated. Ultrasonic waves are applied to the medium in the ultrasonic cleaning tank 43 to clean the surface of the tip 14a of the needle 14. For example, when the surface of the tip 14a of the needle 14 is contaminated with an organic substance or the like, the organic substance is removed. As a result, the surface of the tip portion 14a of the needle 14 is cleaned, and there is no substance that inhibits adsorption of other elements or the like, that is, an active state (Sl).

After the tip 14a of the needle 14 is activated by ultrasonic cleaning, the positioning means 15 is operated to move the tip 14a of the needle 14 into the introduced substance solution 17a injected into a predetermined recess of the container base 17. Is immersed in a predetermined portion of the droplet of the liquid crystal. At this time, a predetermined voltage is applied between the needle 14 and the electrode 46 via the wiring cord 47 for a predetermined time. In general, when the substance to be introduced often has a negative charge and is DNA, the DNA is adsorbed on the surface of the tip 14a of the needle 14 using the needle 14 as an anode (S2). Next, the illumination mode is set to a mode suitable for determining the observation / position data of the operation target (S3).

When the operation target is a cell C which is a biological sample, transmitted light field illumination or phase contrast illumination by the light source 9 is often used as an illumination mode. The biological sample in the sample container 4 is observed under a microscope using bright-field illumination or phase-contrast illumination with the light source 9, and the cell C to be introduced is selected. If necessary, the position data of the selected cell C is stored in the storage unit (position storage means) 12a of the control unit 12 (S4).

Next, the cell C is not adhered and the bottom surface of the sample container 4 is exposed near the cell C to be introduced in the visual field of the microscope. The tip 14a of the needle 14 is lowered and brought close enough within a range where coordinate management can be safely performed (S5). As a means for safely managing coordinates, for example, based on the coordinates of the objective lens 7 at the focus position between the base side of the needle 14 that can be sufficiently observed with a microscope and the bottom surface of the sample container 4, A method of lowering the tip 14a of the needle 14 to a height of about 10 to several tens / zm can be cited.

Next, the illumination mode is switched to total reflection illumination from the direction of falling onto the bottom surface of the sample container 4 using the fluorescent lamp 29 (S6). The total reflection illumination is suitable for detecting the position of the treatment tool. In addition, the position of the eccentric slit 31 is adjusted using the adjusting means 31a, and the upper part of the bottom surface of the sample container 4 is irradiated with so-called pseudo-evanescent light in which some transmitted light is mixed in addition to evanescent light. And At the same time, adjust so that the focus of the microscope is located at 5 to LO / zm above the bottom surface of the sample container 4. Then, for example, the needle 14 is lowered at a minute feed of about 2 m per feed, and when the scattered light of the pseudo evanescent light reflected at the tip 14 a of the needle 14 starts to be detected by the image processing means 11. , Stop the needle 14 from descending. This completes the coarse approach.

Next, as described above, the position of the eccentric slit 31 is adjusted using the adjusting means 31a, so that only the evanescent light is irradiated above the bottom surface of the sample container 4. At the same time, adjust so that the focus of the microscope is located within about 20 Onm above the bottom or the force that matches the bottom of the sample container 4. Then, for example, the needle 14 is lowered by a very small feed of about 100 nm per feed, and the evanescent light reflected by the tip 14a of the needle 14 is moved downward. At the time when the scattered light starts to be detected by the image processing means 11 (that is, when the state where the scattered light is not lit and the force changes to the lit state), the lowering of the needle 14 is stopped. This completes the fine movement approach. The height of the needle 14 at this time is defined as a reference height. That is, the position of the needle 14 when the tip 14a is in the very close position immediately before contacting the bottom surface of the sample container 4 is defined as a reference height, and this value is stored in the form of data in the storage unit 12a in the control unit 12. (S7).

When the scattered light of the evanescent light reflected by the tip 14a of the needle 14 is detected by the image processing means 11, in addition to the height position data of the tip 14a of the needle 14, the horizontal plane of the tip 14a of the needle 14 Position data in the X and Y directions along. Therefore, the detected position data in the XY direction is stored in the storage unit 12a in the control unit 12 as the accurate position of the tip 14a of the needle 14.

As described above, the illumination optical system is set to the first illumination mode for detecting the tip (first region) 14a of the needle 14, and in this state, the needle 14 is focused on the tip 14a. When the tip 14a is detected, the illumination optical system is switched to the second illumination mode for irradiating the cell C (the second area) to be operated with illumination light. Set and detect cell position. As described above, the illumination mode is switched to a mode suitable for detecting the distal end portion 14a of the needle 14 as the treatment tool or a mode suitable for detecting the cell C as the operation target, and the The position and the position of the cell are detected.

In FIG. 6, X indicates the optical path of the total reflection illumination optical system, Y indicates the immersion oil interposed between the objective lens 7 and the bottom surface of the sample container, and Z indicates the evanescent illumination area.

[0062] Next, after the needle 14 is once retracted upward by the driving means 25, the driving means 3a is used so that the tip 14a of the needle 14 is positioned at a predetermined position such as the nucleus of the cell C to be introduced. To move XY stage 3. Then, in this state, the driving means 25 is driven, and the needle 14 is lowered. This operation may be automatically performed by a command signal issued from the control unit 12 based on data input to the control unit 12. Then, the distal end 14a of the needle 14 is inserted into a predetermined position of the cell C to be introduced. At this time, the reaching height of the tip 14a of the needle 14, that is, the penetration limit height is set a predetermined distance (for example: m) above the reference height set in S7, so that the tip 14a of the needle 14 Does not collide with the bottom of sample container 4 Therefore, the tip portion 14a of the needle 14 can be surely penetrated to the target position in the cell C (S8).

The data to be input to the control unit 12 may be the stored tip position data of the needle 14 described above. In this case, the predetermined position of the cell C and the relative position of the needle 14 to the tip portion 14a are obtained. The tip 14a of the needle 14 may be inserted into a predetermined position of the cell C to be introduced by using a method of matching the positions.

After the tip 14a of the needle 14 is inserted into the cell C, the driving means 25 is driven to raise the needle 14 and retreat from the cell C. The above is the step of introducing the substance into the target cell C. As a result, the DNA adsorbed on the needle 14 drops from the needle 14 in the nucleus of the cell C, whereby the gene is transferred.

Further, when introducing into a different cell C, the needle 14 is inserted into the washing unit 16 to wash and remove the introduced substance remaining in the needle 14 and the substance adhered in the cell C. Thereafter, the needle 14 is washed with the washing unit 16 to absorb a predetermined substance to be introduced, and the needle 14 is moved to the biological sample until the cells have been selected and the introduction into all the planned locations is completed. The step of inserting the substance into the cell C by inserting the substance into the predetermined position is repeated (S9).

Note that the procedure for introducing a gene is not limited to the order described above, but may be changed as appropriate in accordance with the purpose of introducing a gene and within the range of maintaining necessary accuracy. For example, the cell position detection (S3, S4) may be performed after the introduction needle position detection (S6, S7), or the cleaning of the needle tip (S1) and the adsorption of DNA (S2) may be performed on the cell or needle tip. After the detection (S3 to S7), the detection may be performed immediately before gene introduction (S8). In addition, when introduction is performed several times in one sample container 4 and the needle is used continuously without replacing the needle each time, the detection of the needle tip position (S6, S7) is performed once at first. May be performed only.

In this embodiment, the needle 14 is gradually lowered by the raising and lowering means including the driving means 25 and the Z stage 22 under observation by the inverted microscope 2 using total reflection illumination in the falling direction. At this time, when the tip 14a of the needle 14 reaches the evanescent illumination area Z, it is illuminated with evanescent light. At this time, the scattered light emitted from the tip 14a of the needle is detected by the image processing means 11, and the height position of the needle 14 at this time is set as the reference height. Then, based on the reference height, the height of the needle 14 when the needle 14 is inserted into the cell C is set. The needle 14 can be safely inserted into the cell C without the tip 14a of the 14 coming into contact with the bottom surface of the sample container 4. In addition, even if the tip 14a of the needle 14 is as fine as V which cannot be captured by an optical microscope, the tip 14a can be prevented from colliding with the bottom surface of the sample container 4 and being broken.

In addition, the retracting / advancing operation of the needle 14 using the driving unit 24, the raising / lowering operation of the needle 14 using the driving unit 25, the moving operation of the XY stage 3 using the driving unit 3a, and the illumination of the inverted microscope 2 The switching operation may be performed automatically based on a command signal from the control unit 12, or may be performed manually. When the above operation is performed manually, after the descending position of the needle 14 after obtaining the reference height reaches the set cell C intrusion limit height, a command signal from the control unit 12 is issued. Based on this, it is also possible to restrict further downward movement of the needle 14 and to display on the display means (not shown) connected to the control unit 12 that the position at the time of the downward movement of the needle 14 has reached a predetermined height. good.

It should be noted that the present invention is not limited to the above-described embodiment, but can be appropriately changed as needed.

For example, in the above-described embodiment, the manipulator device 1 of the present invention and a method of operating a treatment tool using the device are described by taking, as an example, the case of gene transfer using insertion and injection with the needle 14. The force described above may be replaced with another treatment tool instead of the needle 14, and operations such as moving, cutting, holding, and physical or electrical stimulation may be performed. In addition, the above operation may be performed on other biological samples such as bacteria that are not limited to cells C, and on works other than biological samples such as semiconductors and nanotubes.

[0068] In the above-described embodiment, electricity is supplied to the outer peripheral surface of the needle 14 to electrically adsorb the introduced substance. However, the present invention is not limited to this. In addition, if it is sufficiently fine, it may be formed in a nozzle shape, and the introduced substance solution may be directly discharged to the cell C or the like from the tip.

[0069] In the above-described embodiment, the force of cleaning the needle 14 using ultrasonic waves is not limited to this, and plasma, ultraviolet light, ozone, or the like is used according to the purpose or situation. Dry cleaning may be used. Instead of washing and reusing the needles 14, a plurality of needles 14 may be prepared in advance, and the needles may be changed and a predetermined operation may be performed. [0070] In the above-described embodiment, the power using fluorescent lamp 29 as the light source for the total reflection illumination in the incident direction may be used as a light source. Further, the position set in advance as the position of the tip 14a of the needle 14 and stored in the storage unit 12a in the control unit 12 is modified based on the position of the scattered light due to the evanescent light detected by the image processing unit 11. You may correct it.

Next, a second embodiment according to the present invention will be described with reference to FIG.

The manipulator device la shown in FIG. 7 includes a lamp 50, an illumination optical system 50a, a perforated mirror 51, a dark-field objective lens 52, an imaging lens 53, an imaging unit 10, an image processing unit (detection unit). 11). An illumination light guide 52a is provided on the outer periphery of the dark field objective lens 52. The needle 14 is supported by a driving means (first positioning means) 67 for aligning a position where the tip portion 14a can exist with the optical axis of the observation optical system. The illumination optical system of the computer device la is a dark-field illumination optical system.

[0072] The illumination light beam emitted from the lamp 50 enters the perforated mirror 51 via the illumination optical system 50a, and enters the illumination light guide 52a as a ring-shaped light beam. The luminous flux incident on the illumination light guide section 52a is reflected or refracted at the tip of the dark field objective lens 52 as shown by the broken line in the figure, and has an angle larger than the angle of the light beam that can be captured by the objective lens 52. Illuminate sample container 4 in degrees.

Here, the light beam reflected by a relatively large and flat surface such as the bottom surface of the sample container 4 does not enter the objective lens 52, but is reflected in a fine shape like the tip 14a of the needle 14. Some of the scattered light generated as a result includes light incident on the objective lens 52. This light is captured by the objective lens 52, passes through the opening of the perforated mirror 51, is focused by the imaging lens 53, forms an image, and is imaged by the imaging means 10. As a result, the image processing means 11 detects that the scattered light has changed from a non-shining state to a shiny state.

[0074] The dark-field illumination light is partially reflected at the interface between the sample container 4 and the medium Ca, partially refracted, transmitted through the medium Ca, and also illuminates the part remote from the bottom surface of the sample container 4. . Thus, even if the tip 14a of the needle 14 is separated from the bottom surface force of the sample container 4, the tip 14a can be detected by the image processing means 11. For this reason, the possibility of collision or damage of the distal end portion 14a of the needle 14 can be further reduced as compared with the case of illuminating the evanescent light. Also, all Since there is no need for a reflective state, the workability is improved because it is not necessary to use an expensive oil immersion type objective lens.

Next, a third embodiment according to the present invention will be described with reference to FIG.

The manipulator device lb shown in FIG. 8 includes a lamp 50, an illumination optical system 50a, a beam splitter 54, a two-way disk 55, a rotating motor 56, an imaging lens 53, an imaging unit 10, an image Processing means (detection means) 11. As shown in FIG. 9, the two-way disc 55 is provided with a small opening 55a in a snail shape. The distance between the minute openings 55a is about 10 times the diameter of the minute openings 55a. The rotation motor 56 rotates the two-way disc 55 at a constant speed. The needle 14 is supported by driving means (second positioning means) 68 for aligning the position where the tip portion 14a can exist with the optical axis of the observation optical system. In this manipulator device lb, the two-way disk 55 is arranged at the image position of the imaging lens 53, and constitutes a disk scanning confocal microscope together with the illumination optical system and the observation optical system.

The illumination light beam emitted from the lamp 50 is reflected by the beam splitter 54 via the illumination optical system, and is incident on the two-way disk 55. The light beam passing through the minute aperture 55a converges at a predetermined point via the imaging lens 53 and the objective lens 7, and illuminates and reflects the tip 14a of the needle 14 that has reached that point. The light reflected by the distal end portion 14a passes through the objective lens 7 and the imaging lens 53 again and is incident on the portable disk 55. Here, if the position where the tip 14a is illuminated and the micro aperture 55a are optically conjugate, if the tip 14a is in focus, the reflected light (scattered light) will If the tip 14a is not focused on the needle 14a, the reflected light will not converge on the -Po disk 55 and will pass through the aperture 55a in a focused state. Can not. The light that has passed through the small aperture 55a in a converged state passes through the beam splitter 54, forms an image on the imaging unit 10, and forms an image. As a result, the image processing means 11 detects that the reflected light has changed to a non-imaging state and an image forming state.

[0077] Since the two-way disc 55 rotates at a constant speed, the minute aperture 55a can scan the entire focal plane, and only an image of an object on the focal plane is detected. I can't be dumb Therefore, since the image is displayed only when the tip portion 14a of the needle 14 reaches the focal plane, the height of the needle 14 can be detected with high accuracy. Here, the image of the tip 14a of the needle 14 is You can also visually observe with an eyepiece instead of a CCD camera!

Further, as shown in FIG. 10, an upright type microscope may be used as the microscope, and a laser scanning type confocal optical system may be used. The manipulator device lc shown in FIG. 10 includes a laser light source 60, a beam splitter 61, a two-dimensional light operation mechanism 62, a pupil projection lens 63, an imaging lens 53, an objective lens 7, and a condenser lens 64. , A confocal pinhole 65 and a photodetector 66. The two-dimensional optical scanning mechanism 62 uses two sets of galvanomirror scanners to deflect (scan) the laser light in two directions. As the photodetector 66, a photodiode or a photomultiplier (photomultiplier) is employed.

The laser light emitted from the laser light source 60 passes through the beam splitter 61 and enters the two-dimensional optical scanning mechanism 62. The laser light incident on the two-dimensional light scanning mechanism 62 is deflected in two directions by the two-dimensional light scanning mechanism 62, passes through the pupil projection lens 63, the imaging lens 53, the objective lens 7, and the treatment tool such as the needle 14 or a cell. C is irradiated and reflected. The light reflected by the treatment tool such as the needle 14 or the cell C again enters the two-dimensional optical scanning mechanism 62 via the objective lens 7, the imaging lens 53, and the pupil projection lens 63. Then, the deflection in two directions is eliminated, and the light enters the beam splitter 61. The light reflected by the treatment tool such as the needle 14 or the cell C is reflected by the beam splitter 61, collected by the condenser lens 64, and guided to the confocal pinhole 65.

When the treatment tool such as the needle 14 or the cell C is at the focused position of the objective lens 7, the position irradiated with the laser beam and the confocal pinhole 65 are optically conjugated as described above. If so, the convergent light from the condenser lens 64 converges to one point on the confocal pinhole 65 and passes through the confocal pinhole 65. Then, the light is detected by the photodetector 66 and converted into an electric signal. When the treatment tool such as the needle 14 or the cell C is not at the in-focus position of the objective lens 7, the convergent light by the converging lens 64 is not converged on the confocal pinhole 65 but spreads. Even if a part of the light passes through the confocal pinhole 65, it is not detected by the photodetector 66.

When the light detection of the photodetector 66 is performed in synchronization with the light deflection of the two-dimensional optical scanning mechanism 62, the convergent light is only emitted when the treatment tool such as the needle 14 or the cell C has the focus of the objective lens. Imaged. Furthermore, when images are captured while sequentially changing the height of the objective lens, the height between the cell C and the treatment tool 14 depends on the height of the treatment tool such as the needle 14 and the height of the objective lens 7 from which an image of the cell C can be obtained. Can be detected.

In such a manipulator using a disk-type or laser-scanning confocal optical system, it is possible to detect not only the image of the distal end of the treatment tool but also its height with high accuracy. Can be.

Claims

The scope of the claims

[1] A manipulator device that performs an operation on an operation target under observation by an observation optical system:

A treatment tool for performing an operation on the operation target;

A tip included in the treatment tool and in contact with or closest to the operation target; and a change in light including scattered light generated by the illumination light and generated by displacement of the tip. Detection means for detecting

A manipulator device comprising:

[2] The manipulator device according to claim 1, wherein

The illumination optical system further includes an illumination optical system that selectively irradiates the illumination light to an area where the distal end portion may exist, among the areas observed by the observation optical system.

[3] The manipulator device according to claim 2, wherein

The illumination light is radiated through the observation optical system to a region where the distal end may exist.

[4] The manipulator device according to claim 2, wherein

The illumination optical system generates evanescent light on a surface holding the operation target.

[5] The manipulator device according to claim 2, wherein

A first position aligning unit that aligns a position where the distal end can exist with an optical axis of the observation optical system,

The illumination optical system is a dark field illumination optical system.

[6] The manipulator device according to claim 2, wherein

A second alignment unit that adjusts a position where the distal end portion may exist and a focal position of the observation optical system,

The detection unit detects a change in light including the scattered light that has passed through an opening provided at a position optically conjugate to the region irradiated with the illumination light.

[7] The manipulator device according to claim 6, wherein

The observation optical system includes a disk having a plurality of the openings, and By rotating the disk, a region where the tip portion may exist is scanned and illuminated through the opening,

The detecting means detects a change in light including the scattered light that has passed through the opening provided in the disk.

[8] The manipulator device according to claim 6, wherein

The illumination optical system emits laser light, and scans the laser light in a state where the laser light is converged in a region where the front end may exist;

The detecting means detects a change in light including the scattered light passing through the opening provided at a position optically conjugate with the region where the laser light is converged.

[9] The manipulator device according to claim 2, wherein

The illumination optical system includes at least a first illumination mode for irradiating a first area with the illumination light, and a second illumination mode for irradiating a second area different from the first area with the illumination light. Switch between lighting modes.

[10] The manipulator device according to claim 9, wherein

The illumination optical system irradiates the illumination light to a region where the distal end portion may exist in the first illumination mode, and irradiates the operation target with the illumination light in the second illumination mode.

[11] The manipulator device according to claim 1,

The tip is smaller than the optical resolution of the observation optical system.

[12] The manipulator device according to claim 1,

The apparatus further includes position storage means for storing the position of the tip portion detected by the detection means.

[13] The manipulator device according to claim 12, wherein

Driving means for adjusting a relative distance between the surface on which the operation target is held and the treatment tool,

The driving unit controls the movement of the treatment tool based on a result detected by the detection unit.

[14] The manipulator device according to claim 1, The operation with the treatment tool includes inserting the treatment tool into the operation target, injecting a substance into the operation target, cutting the operation target, holding the operation target, and moving the operation target. , And physical or electrical stimulation of the operation target.

[15] The manipulator device according to claim 1,

The operation target includes a biological sample.

[16] The manipulator device according to claim 15, wherein

The biological sample is a living cell.

[17] A manipulator used for the manipulator device according to claim 1.

[18] A method for operating a treatment tool using a manipulator device, which performs an operation on an operation target under observation by an observation optical system, comprising:

A step of illuminating, by illumination light, a tip included in a treatment tool that performs an operation on the operation target and coming into contact with or closest to the operation target;

Detecting a change in light including scattered light of the tip portion illuminated by the illumination light, which is caused by the displacement of the tip portion;

A method for operating a treatment tool using a manipulator device comprising: