JP4331748B2 - Manipulator device and treatment instrument operating method using manipulator device - Google Patents

Description

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 priority about Japanese Patent Application No. 2004-100820 for which it applied on March 30, 2004, and uses the content here.

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 electroporation method or a particle gun method, a calcium phosphate method There are known methods using cell endocytosis, such as lipofection and lipofection.
Among these methods, in the microinjection method described in Patent Document 1 below, a DNA solution is directly injected into individual cells, so that genes can be selectively introduced into arbitrary cells. In this method, if the tip of the treatment tool of the manipulator device is thick, damage to the biological sample increases, and the survival rate after the operation cannot be said to be good, which may hinder the experiment after introduction.
Therefore, for example, in Patent Document 2 below, an ultrafine needle-shaped part that does not cause damage when inserted into a cell is used as a treatment tool, and a gene or a substance involved in gene expression is immobilized on the needle-shaped part. In this state, the gene is introduced into the cell by inserting and retaining the cell.
Japanese Patent Laid-Open No. 06-109979 JP 2003-325161 A

Generally, in a manipulator apparatus for performing gene introduction as described above, selection of target cells is performed under an optical microscope. Similarly, the tip of a treatment tool that treats 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 instrument is reduced in order to reduce damage to cells, it is difficult to detect the distal end of the treatment instrument with an optical microscope. . Therefore, in Patent Document 2, an atomic force microscope (AFM) is added to measure the distance between the treatment tool and the cell, and to measure the atomic force acting between the cantilever that is the treatment tool and the cell. Is going. Therefore, there is a drawback that an atomic force microscope that is expensive and complicated in structure and operation is required.
Further, even with such a configuration, it is not possible to accurately align the cell and the distal end of the treatment instrument.

  The present invention has been made in view of the above problems, and has an object to accurately obtain the position of the distal end portion of the treatment instrument of the manipulator with a low-cost and simple configuration, and to satisfactorily control the position of the distal end portion. And

  The present invention relates to a manipulator device that performs an operation on an operation target under observation by an observation optical system: a treatment tool that performs an operation on the operation target; included in the treatment tool; A tip portion that is in contact with or closest to the object; and detecting means that detects a change in light including scattered light from the tip portion that is illuminated by illumination light and that is generated when the tip portion is displaced. A manipulator device is provided.

  According to the manipulator device described above, even if the treatment tool is very fine and difficult to observe, the treatment tool can be detected only by detecting scattered light from the distal end of the treatment tool and using it. It is possible to accurately determine the position of the distal end portion without touching and without fear of damaging the treatment instrument.

  It is preferable that the manipulator device further includes an illumination optical system that selectively irradiates the illumination light to a region where the tip portion can exist among regions observed by the observation optical system.

  According to the manipulator device described above, the detection means can detect a change in the image of scattered light only when the distal end of the treatment instrument reaches a predetermined region to be positioned, so that the position of the distal end of the treatment instrument is detected. Accuracy can be improved.

  In the manipulator device described above, it is preferable that the illumination light is irradiated to a region where the tip portion can exist through the observation optical system.

  In the manipulator device described above, it is preferable that the illumination optical system generates evanescent light on a surface that holds the operation target.

  According to the manipulator device described above, the distal end portion of the treatment instrument can be brought close to the surface holding the operation target as close as possible to 50 to 150 nm. Since it is detected, the accuracy of detecting the position of the distal end portion of the treatment instrument can be greatly improved.

  The manipulator device may further include a first alignment unit that aligns a position where the tip portion can exist and an optical axis of the observation optical system, and the illumination optical system is a dark field illumination optical system. preferable.

  According to the manipulator device described above, the treatment at a position away from the objective lens without directly detecting the illumination light from the illumination optical system by using the dark field observation optical system and moving the distal end portion of the treatment instrument. Even with a tool, it is possible to selectively detect an image of scattered light from the tip portion with a simple microscope configuration.

The manipulator device is provided at a position optically conjugate with the second alignment means for aligning the position where the tip portion can exist and the optical axis of the observation optical system, and the region irradiated with the illumination light. further comprising a member having an opening portion, said detecting means preferably detects a change in the light including the scattered light that has passed through the opening.

  According to the manipulator device described above, the distal end portion of the treatment tool is moved after using the confocal optical system, so that the portion illuminated by the illumination optical system is treated at a position optically conjugate with the opening. Since the image of the scattered light can be formed on the detection means only when the distal end of the instrument reaches, the scattered light from the distal end of the treatment instrument can be selectively captured, and the distal end of the treatment instrument The accuracy of detecting the position can be greatly improved.

  In the manipulator device, the observation optical system includes a disk having a plurality of openings, and scans and illuminates a region where the tip portion can exist through the opening by rotating the disk. It is preferable that the detection means detects a change in light including the scattered light that has passed through the opening provided in the disk.

  According to the manipulator device described above, by rotating the disk, the illumination light is scanned within the observation visual field, and the scattered light from the distal end portion of the treatment instrument that has reached the in-focus portion of the observation optical system is detected. Can be reliably detected.

  In the 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 tip portion can exist, and the detection unit converges the laser light. 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 with the formed region.

  According to the manipulator device described above, the detection means reliably detects the scattered light from the distal end portion of the treatment instrument that has reached a position optically conjugate with the opening by scanning the laser beam within the observation visual field. Can do.

In the manipulator device described above, the operation target is different from the first region in which the illumination optical system irradiates at least the first region where the tip portion can exist with the illumination light. It is preferable to switch each of the second illumination modes in which the illumination light is irradiated to the second region where an object can exist .

  According to the manipulator device described above, it is possible to perform various operations by switching between a rough position detection and a precise position detection of the distal end of the treatment instrument, illumination on the distal end of the treatment instrument, illumination on other parts, and the like. it can.

In the manipulator device described above, the illumination optical system has at least a first illumination mode in which the illumination light is irradiated on the distal end portion and a second illumination mode in which the operation object is irradiated with the illumination light, respectively. It is preferable to switch .

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

  In the manipulator device, it is preferable that the tip portion is smaller than an optical resolution of the observation optical system.

  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 such a treatment tool can be reliably used as long as it is a method for detecting scattered light. In addition, the position of the tip can be detected.

  The manipulator device preferably further includes a position storage unit that stores the position of the tip portion detected by the detection unit.

  According to the manipulator device described above, if the position of the distal end portion of the treatment instrument is stored as, for example, the reference position, the position control of the distal end portion of the treatment instrument can be reliably performed with one position detection.

  The manipulator device further includes a drive unit that adjusts a relative distance between the surface on which the operation target object is held and the treatment tool, and the drive unit is configured based on a result detected by the detection unit. It is preferable to control the movement of the treatment instrument.

  According to the manipulator device described above, the position of the treatment instrument detected by the detection means is used as a reference position, and the position of the treatment instrument is adjusted and the operation target is operated without damaging the treatment instrument. Can do.

  In the manipulator device, the operation by the treatment tool is performed by inserting the treatment tool into the operation target object, injecting a substance into the operation target object, cutting the operation target object, holding the operation target object, and It is preferable to include physical or electrical stimulation to the operation target.

  In the above-described manipulator device, the operation refers to various manipulations including cases where the distal end portion of the treatment tool touches or does not touch the object.

  In the manipulator device, it is preferable that the operation target includes a biological sample. The biological sample is preferably a living cell.

  The present invention is a treatment tool operation method using a manipulator device that performs an operation on an operation target under observation of an observation optical system, and includes: a treatment tool that operates on the operation target; A step of illuminating the distal end portion that is in contact with or closest to the operation target object with illumination light; and light that includes scattered light from the distal end portion that is illuminated by the illumination light and that is generated when the distal end portion is displaced. And a treatment tool operating method using a manipulator device comprising:

  Technologies such as illumination of minute areas using evanescent light, dark field illumination optical systems, confocal optical systems, etc. have existed conventionally, but the technical idea of detecting very fine needle tips using these technologies is, It did not exist in the past. However, according to the manipulator device described above, even if the treatment tool is difficult to observe, the scattered light from the distal end of the treatment tool is detected, and the treatment tool does not touch the object simply by using it. The position of the tip can be accurately obtained without fear of damaging the treatment tool.

  The illumination optical system that selectively irradiates illumination light to a predetermined region that can be observed by the observation optical system of the present invention includes a surface that holds an operation target and transmits light necessary for observation. It is possible to apply a total reflection illumination optical system that totally reflects at the interface between the substrate to be operated and the medium of the operation target.

  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 its tip enters this region, the evanescent wave reflected by the tip is An image is formed on the detector through the system and detected. The area where the evanescent wave exists, that is, the area illuminated by the total reflection illumination optical system is an area of about 50 to 150 nm, and since only the distal end portion of the treatment instrument is illuminated, only the distal end portion can be detected with good contrast. it can.

  As the illumination optical system, a dark field illumination optical system can also be applied. In the dark field illumination optical system, since the illumination light is illuminated from the outside of the objective lens at an angle larger than the numerical aperture of the objective lens, the light reflected by the horizontal plane does not enter the objective lens directly. However, if a minute object such as the distal end portion of the treatment instrument is present in the illumination region, a part of the reflected light is incident on the objective lens, so that the distal end portion of the treatment instrument can be detected.

  Further, 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 imaging position can be applied. When these are used, only the region in focus on the sample is illuminated and imaged, so that the tip can be detected only when the tip of the treatment tool is in this region. As the confocal optical system, either a disk scanning type or a laser scanning type is applicable.

  Furthermore, the manipulator device according to the present invention further includes a driving unit that adjusts a relative distance between the treatment object or the surface on which the manipulation object is held and the treatment tool, and the driving unit is selected through the observation optical system. The relative distance between the surface on which the operation target is held and the treatment tool may be adjusted based on the image of the treatment tool detected by the detection means that detects the target.

  In such a configuration, the mode for illuminating the surface on which the operation target is held and the mode for illuminating the treatment tool can be switched. And it can detect easily by illuminating the operation target object or the surface on which the operation target object is held, and the distal end of the treatment instrument with an optimal illumination method. Thereby, it becomes possible to adjust the relative distance between the treatment tool and the operation target.

  According to the present invention, it is possible to satisfactorily detect the image of the distal end portion of the treatment instrument. Therefore, even if the treatment tool is so fine that it cannot be captured by a normal optical microscope, the predetermined operation can be safely performed on the operation target without causing the tip of the treatment tool to contact or break the substrate. It can be carried out. Therefore, the insertion depth to the operation target can be accurately managed, and the operation on the operation target can be performed very efficiently.

It is a figure which shows 1st Embodiment of the manipulator apparatus of this invention, Comprising: It is a figure which shows schematic structure of a manipulator apparatus. It is a sectional side view which shows the outline of the total reflection illumination optical system of an inverted microscope. It is sectional drawing which shows the structure of a washing | cleaning unit. It is a side view which shows the structure of a solution stand. It is a flowchart which shows the operation | movement procedure of the manipulator apparatus in embodiment of this invention. It is sectional drawing which shows the relationship between the biological sample and the needle | hook of a total reflection state in an inverted microscope. It is a figure which shows 2nd Embodiment of the manipulator apparatus of this invention, Comprising: It is a sectional side view which shows the outline of the dark field illumination optical system of the inverted microscope with which a manipulator apparatus is equipped. It is a figure which shows 3rd Embodiment of the manipulator apparatus of this invention, Comprising: It is a sectional side view which shows the outline of the confocal optical system of the inverted microscope with which a manipulator apparatus is equipped. It is a schematic diagram of the Nipo disk used for a confocal optical system. It is a figure which shows the modification of the said 3rd Embodiment, Comprising: It is a schematic diagram of the laser scanning type confocal optical system in an erecting microscope.

Explanation of symbols

1, 1a, 1b, 1c: Manipulator device, 2: Inverted microscope, 2a: Total reflection illumination optical system, 3: XY stage, 3a: Driving means, 4: Sample container, 5: Manipulator main body, 6: Main mount, 7 : Objective lens, 8: eyepiece lens, 9: light source, 10: imaging means, 11: image processing means, 12: control unit, 13: holding device, 14: needle, 14a: tip part, 14b: base part, 14c: power supply Code: 15: Positioning means, 16: Cleaning unit, 17: Solution stage, 17a: Introducing substance solution, 21: Retraction stage, 21a: Holding member, 22: Z stage, 24, 25: Driving means, 26: Supporting member, 29: fluorescent lamp, 30: wedge prism, 31: eccentric slit, 31a: adjustment means, 32: excitation filter, 33: mirror, 34: lens, 35: dichroic mirror, 41: cover 41a: upper surface portion, 42: opening, 43: ultrasonic cleaning tank, 44: ultrasonic transducer, 45: wiring cord, 46: electrode, 47: wiring cord, 50: lamp, 51: perforated mirror, 52: Dark field objective lens, 52a: illumination light guide, 53: imaging lens, 54: beam splitter, 55: Nipo 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 alignment) means)

A 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. For example, the manipulator device 1 introduces an appropriate gene (introduced substance) into the nucleus of a cell (biological sample) C that is an operation target existing in the medium Ca, and expresses the protein encoded by the gene. (See FIG. 6).

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

  The inverted microscope 2 includes an XY stage 3 that can be moved in two horizontal directions (front and rear and left and right) by installing a sample container 4 and the like, a main gantry 6 that supports the XY stage 3, and a biological sample in the sample container 4. Objective lens 7 (see FIG. 2) for observing the image from below, an eyepiece lens 8 for observing an image observed by the objective lens 7, and a light source 9 provided on the upper part of the main gantry 6. The inverted microscope 2 is provided with an imaging means 10 including a lens and a CCD camera for taking an image of a biological sample observed with the objective lens 7. 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 3 a 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-rear direction or the left-right direction.

  The manipulator body 5 includes a holding device 13 that holds an introduced substance to be introduced into cells C or the like of a biological sample. The holding device 13 cleans and activates a needle (treatment tool) 14 that is an introduction means inserted into a cell C or the like of a biological sample, a positioning means 15 that controls the position of the needle 14, and a tip 14 a of the needle 14. A cleaning unit 16 to be converted into a liquid, and a solution stage 17 in which an introduction substance to be adsorbed and held by the needle 14 is stored.

  The needle 14 has a sharp tip portion 14a and a base portion 14b having a diameter larger than that of the tip portion 14a. The tip 14 a of the needle 14 is smaller than the optical resolution of the inverted microscope 2. In addition, the needle 14 holds the base portion 14 b in 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 source (not shown) via a power cord 14c. The positioning means 15 includes a retracting stage 21 that holds the base portion 14b of the needle 14, a Z stage 22 that supports the retracting stage 21 so as to be movable in the Z direction (vertical direction), and the XY stage 3. The XY stage 3 is also used as the inverted microscope 2.

The retracting stage 21 has a holding member 21 a that holds the base portion 14 b of the needle 14. The holding member 21 a is connected to the driving unit 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. The Z stage 22 is provided with driving means 25. When the driving unit 25 is driven in accordance with a signal from the control unit 12, the Z stage 22 moves in the vertical direction. That is, the Z stage 22 and the driving unit 25 constitute an elevating unit that raises and lowers the needle 14 via the holding unit 21 a and the retracting stage 21.
The Z stage 22 is attached to the main mount 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 that performs fluorescence observation. The total reflection illumination optical system 2a selectively irradiates illumination light to a region where the distal end portion 14a of the needle 14 can exist among regions observed by the inverted microscope 2, and holds the cell C. The evanescent light is generated on the bottom surface. 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, further reflected by the dichroic mirror 35, and enters the back surface side (lower surface side) of the sample container 4 through the dedicated objective lens 7.

  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 illumination light to the objective lens 7 at least in the vicinity of the critical angle, and hence the incident angle to the back surface of the sample container 4. Note that 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 is substantially rectangular and has a space inside. An opening 42 into which the needle 14 is inserted is provided at the approximate center of the upper surface portion 41 a of the cover 41. The diameter of the opening 42 is such that the needle 14 can be inserted in a necessary amount for dust prevention. An ultrasonic cleaning tank 43 and an ultrasonic vibrator 44 are disposed in the cover 41. The ultrasonic cleaning tank 43 holds a medium (for example, pure water, a neutral detergent, or an alkaline solution) that can sufficiently immerse the tip portion 14a of the needle 14. The ultrasonic transducer 44 is attached to the lower part of the ultrasonic cleaning tank 43. A wiring cord 45 is connected to the ultrasonic transducer 44, and the wiring cord 45 passes through the cover 41 and is connected to a power source (not shown).
In addition, when using a detergent and a chemical | medical agent as a washing | cleaning liquid, neutralization and a rinse are fully performed using the some ultrasonic cleaning tank 43. FIG.

As shown in FIG. 4, the solution table 17 is provided with a plurality of recesses at predetermined intervals. For each of the plurality of recesses, for example, an introduction substance solution 17a such as a plurality of different types of DNA (deoxyribonucleic acid) solutions 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 recess for dispensing the solution 17a in the solution table 17, the upper surface of the solution table 17 is water-repellent and a plurality of hydrophilic regions are formed, and the solution 17a is held in the hydrophilic region. It may be.

Using such a manipulator device 1, a procedure for holding the introduced substance at the distal end portion 14 a of the needle 14 and introducing the introduced substance into the biological sample will be described with reference to the flowchart shown in FIG. 5.
First, the positioning means 15 shown in FIG. 1 is moved, the needle 14 is inserted into the opening 42 of the cleaning unit 16, and the tip end portion 14a of the needle 14 is placed in the ultrasonic cleaning tank 43 as indicated by a virtual line in FIG. Immerse in the medium. In this state, power is supplied from the power source to the ultrasonic transducer 44 via the drive wiring 45 to vibrate the ultrasonic transducer 44. Ultrasonic waves are applied to the medium in the ultrasonic cleaning tank 43, and the surface of the tip end portion 14 a of the needle 14 is cleaned. 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 14a of the needle 14 is cleaned, and a state in which there is no substance that inhibits adsorption of other elements or the like, that is, an active state (S1).

When the tip 14a of the needle 14 is activated by ultrasonic cleaning, the positioning means 15 is activated to cause the tip 14a of the needle 14 to move the droplet of the introduced substance solution 17a injected into a predetermined recess of the container base 17. Insert into a predetermined place and immerse. 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. When the introduced substance is DNA that generally has a negative charge, 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 one suitable for observation / position data determination of the operation target (S3).

  When the operation target is a cell C which is a biological sample, transmitted bright field illumination or phase difference illumination using the light source 9 is often used as the illumination mode. A biological sample in the sample container 4 is observed with a microscope using bright field illumination or phase difference illumination by a light source 9, and a 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 unit) 12a of the control unit 12 (S4).

  Next, in the vicinity of the cell C to be introduced in the microscope field of view, the tip 14a of the needle 14 is connected via the driving means 25 or the like to the location where the cell C is not adhered and the bottom surface of the sample container 4 is exposed. , And approach sufficiently within a range where coordinate management can be safely performed (S5). As means for safely managing the coordinates, for example, 10 coordinates from the bottom surface of the sample container 4 based on the coordinates of the objective lens 7 at the focus position between the base side portion of the needle 14 that can be sufficiently observed with a microscope and the bottom surface of the sample container 4 are used. There is a method in which the tip end portion 14a of the needle 14 is lowered to a height of about tens of μm.

  Next, the illumination mode is switched to total reflection illumination from the incident direction on the bottom surface of the sample container 4 using the fluorescent lamp 29 (S6). Total reflection illumination is suitable for detecting the position of the treatment instrument. Further, the position of the eccentric slit 31 is adjusted using the adjusting means 31a so that a so-called pseudo-evanescent light in which some transmitted light is mixed in addition to the evanescent light is irradiated above the bottom surface of the sample container 4. At the same time, the focus of the microscope is adjusted so as to be located 5 to 10 μm above the bottom surface of the sample container 4. For example, when the needle 14 is lowered by a minute feed of about 2 μm per feed, and the scattered light of the pseudo evanescent light reflected by the tip 14 a of the needle 14 starts to be detected by the image processing means 11, the needle 14 descent is stopped. This completes the coarse motion approach.

Next, in the same manner as described above, the position of the eccentric slit 31 is adjusted using the adjusting means 31 a so that only the evanescent light is irradiated above the bottom surface of the sample container 4. At the same time, the microscope is adjusted so that the focus of the microscope coincides with the bottom surface of the sample container 4 or is within about 200 nm above the bottom surface. Then, for example, when the needle 14 is lowered by a very small feed of about 100 nm per feed, and the scattered light of the evanescent light reflected by the tip 14a of the needle 14 starts to be detected by the image processing means 11 (that is, When the scattered light changes from the non-lighted state to the lighted state), the lowering of the needle 14 is stopped. This completes the tremor approach. The height of the needle 14 at this time is set as a reference height. That is, the position of the needle 14 when the tip 14a is in a very close position immediately before contacting the bottom surface of the sample container 4 is set as a reference height, and this value is stored in the storage unit 12a in the control unit 12 in the form of data. (S7).
When the image processing means 11 detects the scattered light of the evanescent light reflected by the tip portion 14a of the needle 14, in addition to the height position data of the tip portion 14a of the needle 14, the horizontal plane of the tip portion 14a of the needle 14 is used. It is also possible to detect position data in the XY directions along the line. Therefore, the detected position data in the XY directions is stored in the storage unit 12a in the control unit 12 as the accurate position of the distal end portion 14a of the needle 14.

In this way, the illumination optical system is set to the first illumination mode for detecting the tip portion (first region) 14a of the needle 14, and in this state, the tip of the needle 14 is focused on the tip portion 14a. If the part 14a is detected and the tip part 14a is detected, the illumination optical system is set to the second illumination mode for irradiating illumination light to the cell C (second region) that is the object to be operated, and the cell The position of is detected. In this way, the illumination mode is switched to a mode suitable for detecting the distal end portion 14a of the needle 14 serving as the treatment instrument or a mode suitable for detecting the cell C serving as the operation target, and the distal end portion 14a of the needle 14 is detected. Detect position and cell position.
In FIG. 6, X represents the optical path of the total reflection illumination optical system, Y represents immersion oil interposed between the objective lens 7 and the bottom surface of the sample container, and Z represents the evanescent illumination area.

Next, after the needle 14 is once retracted upward by the driving means 25, the XY stage is provided via the driving means 3a 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. 3 is moved. In this state, the driving means 25 is driven to lower the needle 14. 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. And the front-end | tip part 14a of the needle | hook 14 is inserted in the predetermined position of the cell C to be introduced. At this time, by setting the reaching height of the distal end portion 14a of the needle 14, that is, the intrusion limit height, a predetermined distance (for example, 1 μm) above the reference height set in S7, the distal end portion 14a of the needle 14 is moved. Since it does not collide with the bottom face of the sample container 4, the front-end | tip part 14a of the needle | hook 14 can be reliably penetrated to the target position in the cell C (S8).
The data to be input to the control unit 12 may be the above-described stored tip position data of the needle 14, and the relative position of the cell C at this time and the tip 14a of the needle 14 is obtained. The tip portion 14a of the needle 14 may be inserted into a predetermined position of the cell C to be introduced using a method for matching the two.

  After the distal end portion 14a of the needle 14 is inserted into the cell C, the driving means 25 is driven to raise the needle 14 and retract from the cell C. The above is the step of introducing the substance into the target cell C. As a result, the DNA introduced into the needle 14 is dropped from the needle 14 in the nucleus of the cell C, whereby gene introduction is performed.

Further, when introduction into a different cell C is performed, the needle 14 is inserted into the washing unit 16, and the introduced substance remaining on the needle 14 and the substance adhered in the cell C are washed away. Thereafter, until the cells are selected and the introduction to all the planned locations is completed, the needle 14 is washed by the washing unit 16 to adsorb a predetermined introduced substance, and then the needle 14 is removed from the biological sample. The process of introducing the substance to be introduced into the cell C is repeated (S9).
Note that the procedure for introducing a gene is not limited to the order described above, and may be appropriately changed within a range that matches the purpose of gene introduction and maintains necessary accuracy. For example, the position detection (S3, S4) of the cell may be performed after the position detection (S6, S7) of the introduction needle, or the cleaning of the needle tip (S1) or the adsorption of DNA (S2) is performed by the cell or the needle tip. After detection (S3 to S7), it may be performed immediately before gene introduction (S8). Further, when introduction is performed a plurality of times in one sample container 4 and the needles are used continuously without changing the needle every time, the detection of the needle tip position (S6, S7) is performed once at the beginning. You may just do it.

  In this embodiment, the needle 14 is gradually lowered by the lifting / lowering means including the driving means 25 and the Z stage 22 under observation using the total reflection illumination in the incident direction by the inverted microscope 2. At that time, when the distal end portion 14a of the needle 14 reaches the evanescent illumination area Z, the needle 14 is illuminated with evanescent light. At this time, since the scattered light emitted from the tip 14a of the needle is detected by the image processing means 11, the height position of the needle 14 at this time is set as a reference height. Since the height of the needle 14 when the needle 14 is inserted into the cell C is set based on this reference height, the tip 14a of the needle 14 does not contact the bottom surface of the sample container 4, and the needle 14 14 can be safely inserted into cell C. Even if the tip 14a of the needle 14 is so fine that it cannot be captured by an optical microscope, it can be prevented from colliding with the bottom surface of the sample container 4 and breaking.

Further, 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 switching operation of the inverted microscope 2 are performed. The 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 descent position of the needle 14 after obtaining the reference height reaches the set entry height of the cell C, a command signal from the control unit 12 is output. On the basis of this, the further lowering of the needle 14 is restricted, and a display means (not shown) connected to the control unit 12 displays that the position when the needle 14 is lowered reaches a predetermined height. good.
The present invention is not limited to the above-described embodiment, and can be appropriately changed as necessary.

  For example, in the above-described embodiment, the manipulator device 1 of the present invention and the operation method of the treatment tool using the device have been described by taking the case of gene introduction using insertion / injection with the needle 14 as an example. However, the present invention is not limited to this, and other treatment tools may be used in place of the needle 14 to perform operations such as movement, cutting, holding, physical or electrical stimulation. Further, the operation target is not limited to the cell C, and the above-described operation may be performed on a biological sample such as bacteria or a workpiece other than a biological sample such as a semiconductor or a nanotube.

  In the above-described embodiment, electricity is caused to flow on the outer peripheral surface of the needle 14 to electrically adsorb the introduced substance. However, the present invention is not limited to this, and it may be chemically adsorbed. Further, if it is sufficiently fine, the introduced substance solution may be directly discharged from the tip of the nozzle to the cell C or the like as a nozzle.

  In the above-described embodiment, the needle 14 is cleaned using ultrasonic waves. However, the present invention is not limited to this, and dry cleaning using plasma, ultraviolet light, ozone, or the like is not limited to this. It may be used. Instead of cleaning the needle 14 and reusing it, a plurality of needles 14 may be prepared in advance, and a predetermined operation may be performed by changing the needles.

  In the above-described embodiment, the fluorescent lamp 29 is used as the light source for the total reflection illumination in the epi-illumination direction, but the present invention is not limited to this, and a laser may be used as the light source. Further, the position set in advance as the position of the tip end portion 14a of the needle 14 and stored in the storage unit 12a in the control unit 12 is corrected based on the position of the scattered light by the evanescent light detected by the image processing means 11. May be.

Next, a second embodiment according to the present invention will be described with reference to FIG.
A manipulator device 1a 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 means 10, and an image processing means (detection means). 11. An illumination light guide 52 a is provided on the outer periphery of the dark field objective lens 52. The needle 14 is supported by a driving unit (first positioning unit) 67 that aligns the position where the distal end portion 14a can exist with the optical axis of the observation optical system. The illumination optical system of the manipulator device 1a is a dark field illumination optical system.

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

  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 by a fine shape like the tip portion 14a of the needle 14. Some of the scattered light is 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 condensed by the imaging lens 53, forms an image, and is imaged by the imaging unit 10. Thereby, it is detected by the image processing means 11 that the scattered light has changed from a non-lighted state to a lighted state.

  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 away from the bottom surface of the sample container 4. Thereby, even if the tip end portion 14 a of the needle 14 is separated from the bottom surface of the sample container 4, the tip end portion 14 a can be detected by the image processing means 11. For this reason, the possibility of collision / breakage of the tip end portion 14a of the needle 14 can be further reduced as compared with the case where the evanescent light is illuminated. Further, since a total reflection state is not required, it is not necessary to use an expensive oil immersion type objective lens, and workability is improved.

Next, a third embodiment according to the present invention will be described with reference to FIG.
The manipulator device 1b shown in FIG. 8 includes a lamp 50, an illumination optical system 50a, a beam splitter 54, a Nipo disk 55, a rotation motor 56, an imaging lens 53, an imaging means 10, an image processing means ( Detection means) 11. As shown in FIG. 9, the Nipo disk 55 has a spiral minute opening 55a. The distance between each minute opening 55a is about 10 times the diameter of the minute opening 55a. The rotation motor 56 rotates the Nipo disk 55 at a constant speed. The needle 14 is supported by a driving means (second positioning means) 68 that matches the position where the tip end portion 14a can exist with the optical axis of the observation optical system. In this manipulator device 1b, the Nipo disk 55 is disposed 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 through the illumination optical system and is incident on the Nipo disk 55. The light beam that has passed through the minute aperture 55a passes through the imaging lens 53 and the objective lens 7 and converges at a predetermined point, and illuminates and reflects the distal end portion 14a of the needle 14 that reaches that point. The light reflected by the tip end portion 14 a enters the Nipo disk 55 again through the objective lens 7 and the imaging lens 53. Here, if the position where the tip end portion 14a is illuminated and the minute opening 55a are optically conjugate, the reflected light (scattered light) is on the Nipo disk 55 when the tip end portion 14a is in focus. However, if the needle tip 14a is not in focus, the reflected light will not be collected on the Nipo disk 55 and will pass through the micro opening 55a in a converged state. Can not. The light that has passed through the minute aperture 55a passes through the beam splitter 54, forms an image on the imaging means 10, and is imaged. As a result, the image processing means 11 detects that the reflected light has changed from a state where it is not imaged to a state where it is imaged.

  Since the Nipo disk 55 rotates at a constant speed, the minute aperture 55a can scan the entire focal plane, and only the image of the object at the focal plane is detected, and the non-focal plane is not imaged. Therefore, since the image is formed only when the distal end 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 portion 14a of the needle 14 may be visually observed with an eyepiece instead of the CCD camera.

  Further, as shown in FIG. 10, an erecting type microscope may be used and a laser scanning type confocal optical system may be used. A manipulator device 1c 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, a condenser lens 64, A confocal pinhole 65 and a photodetector 66 are provided. The two-dimensional light scanning mechanism 62 includes two sets of galvanometer mirror scanners, and deflects (scans) the laser light in two directions. For the light detector 66, a photodiode or a photomultiplier (photomultiplier) is employed.

  Laser light emitted from the laser light source 60 passes through the beam splitter 61 and enters the two-dimensional light 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, and passes through the pupil projection lens 63, the imaging lens 53, and the objective lens 7, or a treatment instrument such as the needle 14 or cell C. Is irradiated and reflected. The light reflected by the treatment instrument such as the needle 14 or the cell C enters the two-dimensional light scanning mechanism 62 again through the objective lens 7, the imaging lens 53, and the pupil projection lens 63. Then, the deflection in the two directions is eliminated and the light enters the beam splitter 61. The light reflected by the treatment instrument 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 instrument such as the needle 14 or the cell C is at the in-focus position of the objective lens 7, the position irradiated with the laser light and the confocal pinhole 65 are optically conjugate as described above. The convergent light from the condenser lens 64 converges at one point on the confocal pinhole 65 and passes through the confocal pinhole 65. Then, it is detected by the photodetector 66 and converted into an electric signal. Further, when the treatment tool such as the needle 14 or the cell C is not in the in-focus position of the objective lens 7, the convergent light from the condenser lens 64 spreads on the confocal pinhole 65 without converging. Even if a part passes through the confocal pinhole 65, it is not detected by the photodetector 66.

  When light detection by the light detector 66 is performed in synchronization with the light deflection of the two-dimensional light scanning mechanism 62, the convergent light is imaged only when the treatment instrument such as the needle 14 or the cell C has the focus of the objective lens. The Further, when an image is captured while sequentially changing the height of the objective lens, the height of the cell C and the treatment tool 14 is changed according to the height of the treatment tool such as the needle 14 and the objective lens 7 from which an image of the cell C is obtained. Differences can be detected.

In a manipulator device employing such a disk type or laser scanning type confocal optical system, an image of the distal end portion of the treatment instrument can be detected well, and its height can also be detected with high accuracy. .

Claims (17)

A manipulator device that operates an operation target under observation of an observation optical system:
A treatment instrument for operating the operation target;
A tip included in the treatment tool and in contact with or closest to the operation object;
Detecting means for detecting a change in light including scattered light from the tip portion illuminated by illumination light, which is generated with displacement of the tip portion;
A manipulator device comprising: The manipulator device according to claim 1,
An illumination optical system that selectively irradiates the illumination light to a region where the tip portion can exist among the regions observed by the observation optical system is further provided. The manipulator device according to claim 2,
The illumination light is irradiated to a region where the tip portion can exist through the observation optical system. The manipulator device according to claim 2,
The illumination optical system generates evanescent light on a surface that holds the operation target. The manipulator device according to claim 2,
A first alignment means for aligning the position where the tip portion can exist and the optical axis of the observation optical system;
The illumination optical system is a dark field illumination optical system. The manipulator device according to claim 2,
Second alignment means for aligning the position where the tip portion can exist and the optical axis of the observation optical system, and an opening provided at a position optically conjugate with the region irradiated with the illumination light Further comprising a member ,
The sensor detects a change in the light including the scattered light that has passed through the opening. The manipulator device according to claim 6, wherein
The observation optical system includes a disk having a plurality of openings, and scans and illuminates a region where the tip can exist through the opening by rotating the disk.
The detection means detects a change in light including the scattered light that has passed through the opening provided in the disk. 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 tip portion can exist,
The detection means detects a change in light including the scattered light that has passed through the opening provided at a position optically conjugate with a region where the laser light is converged. The manipulator device according to claim 2,
The illumination optical system includes at least a first illumination mode for irradiating the illumination light to a first region where the tip portion may exist, and a first object where the operation object different from the first region may exist . The second illumination mode for irradiating the illumination light to the second area is switched. The manipulator device according to claim 2,
The illumination optical system includes at least a first illumination mode for irradiating the tip portion with the illumination light, and a second illumination mode for irradiating the operation target different from the first region with the illumination light. Switch each. The manipulator device according to claim 1,
The tip portion is smaller than the optical resolution of the observation optical system. The manipulator device according to claim 1,
The apparatus further includes position storage means for storing the position of the tip detected by the detection means. The manipulator device according to claim 12, wherein
Drive means for adjusting the relative distance between the surface on which the operation object is held and the treatment tool;
The drive means controls the movement of the treatment tool based on the result detected by the detection means. The manipulator device according to claim 1,
The operation by the treatment tool includes insertion of the treatment tool into the operation object, injection of a substance into the operation object, cutting of the operation object, holding of the operation object, movement of the operation object, And physical or electrical stimulation to the manipulation object. The manipulator device according to claim 1,
The operation target includes a biological sample. The manipulator device according to claim 15,
The biological sample is a living cell. A treatment instrument operating method using a manipulator device that operates an operation target under observation of an observation optical system:
A step of illuminating, with illumination light, a distal end portion that is included in a treatment instrument that performs an operation on the operation target and is in contact with or closest to the operation target;
Detecting a change in light including scattered light from the tip illuminated by the illumination light, which occurs with displacement of the tip;
A treatment instrument operating method using a manipulator device comprising:

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