JP2005335020A - Micro object machining device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a micro object machining device of high versatility widely usable for extensive purposes, mainly for application to cells while improving the efficiency of operation processing considerably by changing of operation accuracy and strength of force applied to a micro object to be treated such as the cell, according to the scene of operation. <P>SOLUTION: The micro object machining device is provided with: a first laser light source for outputting ultrashort pulse laser for operating a micro object included in a sample cell by shock wave; a second laser light source for outputting trapping laser for operating the micro object with radiation pressure; and a light guide means for guiding the laser beams outputted from the first and second laser light sources, into the sample cell. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an apparatus for processing a minute object such as a cell, and more particularly to an apparatus for processing a cell or the like using a laser manipulation technique.

Conventionally, a precise manipulation of a minute object such as a cell is generally performed by a mechanical manipulator such as a micropipette equipped with a thin metal needle or a glass tube.
However, the operation of manipulating minute objects such as cells using a mechanical manipulator is a task that requires great skill and patience, and often damages cells and the like in the course of operation.

As a technique created to solve the problems of such a mechanical manipulator, there is a laser manipulation technique for manipulating minute objects such as cells using the radiation pressure of laser light.
According to the laser manipulation technology, it is possible to remotely control a minute object to be manipulated in a non-contact manner using the capture force of the light radiation pressure. Operation (capture, transfer, etc.) can be performed with high precision, and application as a micro object manipulation tool in a wide range of fields centering on the biotechnology field is greatly expected.

  Thus, many laser manipulation devices using such laser manipulation technology have been proposed (see, for example, Patent Document 1).

JP 2003-94396 A

  However, although the conventional laser manipulation device is certainly superior in that it can operate a minute object to be operated with high accuracy without contact, it uses the radiation pressure of laser light. Only a very small force (˜pN) could be applied to the object. For this reason, for example, animal cells attached on the substrate cannot be transported by a temporary foot, and the operable micro objects are limited, and the application range is limited.

In view of the above situation, in recent years, ultrashort pulse lasers such as femtosecond lasers have attracted attention in laser manipulation technology.
Since the femtosecond laser has a very small pulse width of several femtoseconds to several hundreds of picoseconds, the instantaneous arrival power in one pulse is extremely high. For this reason, it is possible to generate a local shock wave by a non-linear phenomenon using multiphoton absorption at a localized location of a transparent or low-absorbing substance. In recent years, however, attempts have been made to apply this to the biotechnology field.

However, attempts currently being made are merely attempts to replace the capturing lasers (such as near-infrared lasers) used in conventional laser manipulation devices with ultrashort pulse lasers.
If a laser manipulation device using an ultra-short pulse laser is used, operations such as cell transfer that were impossible with conventional laser manipulation devices using near-infrared lasers, etc. are possible. 10 μm) operation is required, or when operation with a weak force is required.
Further, in a laser manipulation device using an ultrashort pulse laser, it is difficult to manipulate an object of several millimeters that is possible with a mechanical manipulator.

  In fact, in the manipulation of micro objects, especially cells, strong force is not required in a series of operation processing, but ultra-high accuracy is required, ultra-high accuracy is not required, but relatively high accuracy and slightly strong force are required. There are many cases in which the strength of the force and the accuracy of the operation must be properly used depending on the scene, such as a scene where high precision is required but high precision is not required. However, there was no highly versatile manipulation device that could meet the requirements simultaneously.

  The present invention has been made in view of the above circumstances, and it is possible to change the strength of the force to be applied to a minute object to be processed such as a cell or the operation accuracy according to the scene of the operation. It is an object of the present invention to provide a highly versatile micro object processing apparatus that can greatly improve the efficiency of processing and can be widely used for a wide range of applications centering on application to cells.

The invention according to claim 1 is a first laser light source that outputs an ultrashort pulse laser for operating a minute object contained in a sample cell by a shock wave, and a trapping laser for operating the minute object by radiation pressure. And a light guiding means for guiding the laser light output from the first and second laser light sources into the sample cell. .
The invention according to claim 2 comprises a first laser light source that outputs an ultrashort pulse laser for operating a minute object contained in a sample cell by a shock wave, and a mechanical manipulator for operating the minute object. The present invention relates to a minute object processing apparatus.
The invention according to claim 3 relates to the apparatus for processing a micro object according to claim 1, further comprising a mechanical manipulator for operating the micro object included in the sample cell.

The invention according to claim 4 is characterized in that the light guiding means guides laser beams output from the first and second laser light sources coaxially into the sample cell. It relates to a processing apparatus.
According to a fifth aspect of the present invention, there is provided a laser beam separating means for separating the laser beam output from the first laser light source into a plurality of beams, and the separated laser beam is independently controlled to be led into the sample cell. The micro object processing apparatus according to claim 1, further comprising a control mirror.

The invention according to claim 6 is a first laser light source that outputs an ultrashort pulse laser, and irradiates the ultrashort pulse laser to a plurality of locations in the vicinity of a minute object included in a sample cell, and the minute object is generated by a shock wave. The present invention relates to a micro object processing apparatus characterized by comprising a capturing means for capturing an object.
The invention according to claim 7 is characterized in that the capturing means comprises an electronically controlled mirror that scans the laser light output from the first laser light source along the periphery of the minute object. The present invention relates to a micro object processing apparatus.
The invention according to claim 8 is characterized in that the capturing means comprises a digital micromirror device that simultaneously condenses the laser light output from the first laser light source at a plurality of locations near the periphery of the minute object. The present invention relates to a minute object processing apparatus according to claim 6.

The invention according to claim 9 is configured such that the sample cell is placed on a stage of a microscope, and a transmission image of light irradiated from the light source of the microscope and passed through the sample cell is captured by an imaging device. The present invention relates to a minute object machining apparatus according to any one of claims 1 to 8.
The invention according to claim 10 relates to the apparatus for processing a micro object according to any one of claims 1 to 9, wherein a pulse width of the ultrashort pulse laser is several femtoseconds to several hundred picoseconds.
The invention according to claim 11 is characterized in that the micro object is any one of an intracellular organelle, a cell, a living tissue, a living organ, a protein crystal, or a complex thereof. The present invention relates to the described minute object processing apparatus.

  The micro object processing apparatus according to the present invention can change the strength of the force applied to the micro object to be processed such as cells and the operation accuracy according to the scene of the operation, and greatly improve the efficiency of the operation process. And a highly versatile micro object processing apparatus that can be widely used for a wide range of applications centering on application to cells.

DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, a preferred embodiment of a minute object processing apparatus according to the invention will be described with reference to the drawings.
FIG. 1 is a schematic view showing an example (first embodiment) of a basic configuration of a minute object processing apparatus according to the present invention.
The micro object processing apparatus according to the present invention shown in FIG. 1 includes a first laser light source (1) that outputs an ultrashort pulse laser for operating a micro object contained in a sample cell by a shock wave, A second laser light source (2) for outputting a trapping laser for manipulating a minute object.
As the micro object to be manipulated in the present invention, for example, any one of intracellular organelles, cells, living tissues, living organs, protein crystals, or a complex thereof can be mentioned as a suitable example.

The pulse width of the ultrashort pulse laser output from the first laser light source (1) is several femtoseconds to several hundred picoseconds. As such an ultrashort pulse laser, a picosecond laser or a femtosecond laser is preferably used, and a femtosecond laser is particularly preferably used.
As a specific example of the ultrashort pulse laser output from the first laser light source (1), a femtosecond titanium sapphire laser (800 nm, 120 fs) can be given as a representative example, but the invention is not limited to this. Absent.

As the trapping laser output from the second laser light source (2), a near infrared light laser composed of a semiconductor laser, a YAG laser or the like is preferably used.
This is because when a cell is manipulated as a minute object, near-infrared light is not absorbed by the cell, and operations such as capture and movement can be performed without damaging the cell.
As a specific example of the trapping laser output from the second laser light source (2), Nd ³⁺ : YAG Laser (CW 1064 nm, 1 W) can be given as a representative example, but is not limited thereto. .

In FIG. 1, the first laser light source (1) and the second laser light source (2) described above are incorporated in an inverted microscope (3).
The inverted microscope (3) is capable of moving in the XY direction, and has an electric stage (5) for placing a sample cell (4) such as a cell growth basket, and a position directly below the stage (5). An objective lens (6) that is arranged in the unit and collects the laser light output from the first laser light source (1) and the second laser light source (2) and guides it to the sample cell (4); 6) A light source lamp (7) that irradiates light to a sample cell (4) that is disposed vertically above the stage (5), and a sample cell (4) that is irradiated from the light source lamp (7). An imaging device (8) that captures a transmission image of visible light that has passed through ().

A dichroic mirror (9) and a total reflection mirror (10) are arranged vertically below the objective lens (6) of the microscope (3), and the visible light reflected by the total reflection mirror (10) is imaged (8). Is configured to detect.
The imaging device (8) is composed of a CCD camera, a CMOS camera or the like, and the visible light that has passed through the sample cell (4) passes through a dichroic mirror (9) disposed vertically below the objective lens (6). The light is reflected by the total reflection mirror (10), changed in its direction to a right angle, and incident on the image pickup device (8).

The light guide means for guiding the laser light output from the first laser light source (1) to the dichroic mirror (9) disposed in the inverted microscope (3) includes an electronic control mirror (11) and a dichroic mirror (12). The laser light output from the first laser light source (1) is reflected by the electronic control mirror (11), changed direction and guided to the dichroic mirror (12), and then the dichroic mirror. At (12), the direction is changed and guided to the dichroic mirror (9).
As the electronically controlled mirror (11), a galvano mirror, a piezo-driven mirror, an actuator-driven mirror, or the like is used as appropriate.
On the other hand, the laser beam output from the second laser light source (2) passes through the dichroic mirror (12) and goes straight as it is, and is guided to the dichroic mirror (9).

The laser light from the first laser light source (1) reflected by the dichroic mirror (12) and the laser light from the second laser light source (2) that has passed through the dichroic mirror (12) are coaxially connected to the dichroic mirror (9 Then, the light is further collected by the objective lens (6) coaxially and guided to the sample cell (4).
In this way, by operating two types of laser light coaxially into the microscope and guiding them to the sample cell (4), it is possible to manipulate the cells in the sample cell (4) very easily and efficiently. It becomes.

For operations that require ultra-high accuracy (<10 μm) with a weak force (up to pN), a trapping laser output from the second laser light source (2) is used, and a high force (nN to μN) is used. For operations that require accuracy (<100 μm), an ultrashort pulse laser output from the first laser light source (1) can be used.
Therefore, for example, a series of processing operations are continuously performed with one apparatus, such as isolating sperm cells from a tissue using an ultrashort pulse laser and then transferring the isolated sperm cells using a trapping laser. Therefore, the operation can be performed with high accuracy and high efficiency.

The electronic control mirror (11) is connected to the computer (13), and the laser beam outputted from the first laser light source (1) is changed by changing the angle based on the control processing signal from the computer (13). Are configured to scan.
The computer (13) controls the electronic control mirror (11) by capturing an image picked up by the image pickup device (8), obtaining coordinates of the minute object present in the captured image, and calculating the calculated minute object. The optimal path from the coordinates to the target coordinates of the transfer destination of the minute object is calculated, the movement destination coordinates as the movement destination of the minute object at the next time are calculated from the preset amount of movement, and further at the next time The angle of the electronic control mirror is calculated so that a minute object is guided to the moving coordinates, and a control signal is sent to the electronic control mirror (11) so that the calculated angle matches the actual angle.
The minute object under the microscope is captured by the imaging device (8) as a microscope transmission image, captured in real time by the computer (13), and transferred under the control of the electronic control mirror (11).

A mechanical manipulator (14) is arranged on the stage (5) of the inverted microscope (3).
This mechanical manipulator (14) is a known one such as a micropipette equipped with a thin metal needle or a glass tube, and is used to apply a machine to a minute object in a sample cell (4) placed on a stage (5). It can be operated with a certain force. This operation can be performed on a minute object in the same sample cell (4) placed on the stage (5) in combination with one or both of the two types of laser manipulators. it can.

By providing a mechanical manipulator (14) in addition to the two types of laser manipulators described above, a micro object can be manipulated with a force (> mN) stronger than a laser manipulator, although it is less accurate (<mm). It is also possible.
Therefore, by appropriately using the above-mentioned two types of laser manipulators and mechanical manipulators (14), the strength of the force acting on the minute object to be processed such as cells and the operation accuracy are changed according to the scene of the operation. Therefore, the efficiency of the operation process can be greatly improved, and a highly versatile micro object processing apparatus that can be widely used for a wide range of applications centering on application to cells.

FIG. 2 is a schematic view showing a modified example (second embodiment) of the basic configuration of the minute object processing apparatus according to the present invention.
The apparatus according to this modified example is different from the apparatus shown in FIG. 1 in that the apparatus includes laser light separating means (15) for separating the laser light output from the first laser light source (1) into a plurality of pieces. And an electric mirror unit (16) comprising a plurality of electronically controlled mirrors that independently control the separated laser beams and guide them into the sample cell (4) instead of one electronically controlled mirror (11). Since the other configurations are the same, the same reference numerals are given and redundant description is omitted.

  The laser beam separating means (15) separates the laser beam output from the first laser light source (1) into two, and the laser beams separated into these two beams are independent by different electric mirror units (16). And is guided coaxially with the laser beam from the second laser light source (2) that has passed through the dichroic mirror (12) to the dichroic mirror (9), and further coaxially with the objective lens (6). The light is collected and guided to the sample cell (4).

  In this way, by separating the laser light output from the first laser light source (1) into two and making the laser light separated into these two independently controllable, An operation of using the other laser beam for holding and fixing a plurality of minute objects can be performed, and the operation performance is extremely excellent.

Moreover, in this invention, the apparatus provided with the two operation means of the operation means by the ultrashort pulse laser output from the 1st laser light source (1) demonstrated above, and the operation means by the mechanical manipulator (14), You can also
Such an apparatus can be configured, for example, by removing the second laser light source (2) from the apparatus having the configuration shown in FIGS.

FIG. 3 is a schematic view showing another modified example (third embodiment) of the basic configuration of the minute object processing apparatus according to the present invention.
The apparatus according to this modified example includes a first laser light source (1) that outputs an ultrashort pulse laser, and guides the ultrashort pulse laser to a plurality of locations in the vicinity of a minute object included in a sample cell, and generates shock waves. A capturing means for capturing the minute object is provided.

The pulse width of the ultrashort pulse laser output from the first laser light source (1) is several femtoseconds to several hundreds of picoseconds, as in the first embodiment. As such an ultrashort pulse laser, a picosecond laser or a femtosecond laser is preferably used, and a femtosecond laser is particularly preferably used.
As a specific example of the ultrashort pulse laser output from the first laser light source (1), a femtosecond titanium sapphire laser (800 nm, 150 fs, 20 Hz, 10 mW) can be given as a representative example.

In FIG. 3, the first laser light source (1) described above is incorporated in an upright microscope (3).
The upright microscope (3) is capable of moving in the X-Y direction, and includes an electric stage (5) for placing a sample cell (4) such as a cell growth basket, and the stage (5). An objective lens (6) arranged immediately above and collecting the laser light output from the first laser light source (1) and leading it to the sample cell (4), and arranged vertically below the objective lens (6) The light source lamp (7) for irradiating light to the sample cell (4) placed on the stage (5) and the visible light irradiated from the light source lamp (7) and passed through the sample cell (4). An image pickup device (8) for picking up a transmission image is provided.

A dichroic mirror (9) is arranged vertically above the objective lens (6) of the microscope (3).
The imaging device (8) is composed of a CCD camera, a CMOS camera, or the like, and the visible light that has passed through the sample cell (4) passes through a dichroic mirror (9) disposed vertically above the objective lens (6). The light enters the imaging device (8).

The laser beam output from the first laser light source (1) sequentially passes through the mechanical shutter (20), the 2 / λ plate (21), and the polarizer (22), and then is an electric mirror unit comprising a plurality of electronically controlled mirrors. (16) leads to the dichroic mirror (9) arranged in the microscope (3).
The plurality (two) of electronic control mirrors constituting the electric mirror unit (16) can be controlled independently. One of the scanning directions in the sample cell (4) is the X-axis direction and the other is the Y-axis. Responsible for axial scanning.
As the electronically controlled mirror, a galvano mirror, a piezo-driven mirror, an actuator-driven mirror, or the like is appropriately selected.

The laser light from the first laser light source (1) reflected by the dichroic mirror (12) is guided to the dichroic mirror (9), and then condensed by the objective lens (6) to the sample cell (4). It is guided.
The drive control of the electronic control mirror is performed based on the control processing signal from the computer (13), as described in the first embodiment, and the minute object under the microscope is captured as a microscope transmission image. The image is taken by the device (8), captured in real time by a computer, and transferred under the control of an electronically controlled mirror.

The electric mirror unit (16) can scan the laser light output from the first laser light source (1) along the periphery of the minute object in the sample cell (4) by driving individual electronic control mirrors. Thus, the minute object can be captured.
That is, by scanning the ultrashort pulse laser output from the first laser light source (1) at high speed along the periphery of the minute object, and generating shock waves at short intervals at a plurality of locations near the periphery of the minute object, The minute object is captured in a state of being confined by a shock wave generated from the surroundings.
The time interval of the shock wave generated in the vicinity of the minute object can be set by the gate time of the mechanical shutter (20) (for example, 50 ms), while the scanning speed controls the driving of the electric mirror unit (16). Therefore, by setting the gate time and the scanning speed appropriately, the minute object can be surely confined by the shock wave from the surroundings.
Then, by moving the electric stage (5) in a state where the minute object is captured, the minute object such as a cell can be moved in the sample cell.

  FIG. 4 is a diagram schematically showing a method of moving a minute object by the method as described above, and an ultrashort pulse laser (for example, femtosecond laser) (L2) output from the first laser light source (1). ) Is scanned along the circumference of the minute object (M) as indicated by the arc-shaped arrow in the figure, and shock waves are generated at a plurality of locations (indicated by black dots on the arrow) near the circumference of the minute object, A minute object can be captured by the generated shock wave. Then, by moving the electric stage (5) in this state, the minute object can be moved in the sample cell as indicated by a straight arrow.

  In the apparatus of the third embodiment, the shock wave of the ultrashort pulse laser output from the first laser light source (1) is used to store the sample cell (4) as described in the first embodiment. It is also possible to transfer micro objects and to isolate cells.

FIG. 5 is a schematic view showing still another modified example (fourth embodiment) of the basic configuration of the minute object processing apparatus according to the present invention.
Similar to the apparatus of the third embodiment described above, the apparatus according to this modified example includes a first laser light source (1) that outputs an ultrashort pulse laser and a minute object that includes the ultrashort pulse laser in a sample cell. A capturing means is provided that guides to a plurality of locations in the vicinity of the object and captures the minute object by a shock wave.

Hereinafter, regarding the apparatus according to the fourth embodiment, the same components as those of the third embodiment will be denoted by the same reference numerals and redundant description will be omitted, and only different components will be described.
In the apparatus of the fourth embodiment, the laser light output from the first laser light source (1) sequentially passes through the mechanical shutter (20), 2 / λ plate (21), and polarizer (22), and is projected by the projection lens. After the diameter is expanded, it is guided to the digital micromirror device (24) through the mirror (23).

The digital micromirror device (DMD) (24) is composed of hundreds of thousands of micromirrors (17) of several microns that can be independently driven on a CMOS semiconductor. This is an optical semiconductor device that can freely control the shape and reflectivity of the reflecting surface by being changed by digital control.
The individual micromirrors of the digital micromirror device (24) are controlled by capturing an image picked up by the image pickup device (8) on the computer (13) in real time, and the position of the minute object existing in the captured image. This can be done by obtaining coordinates, determining the laser beam condensing coordinates from the position coordinates, and determining the mirror angle from the determined condensing coordinates.

  As shown in the schematic diagram in the enlarged circle, the laser light guided to the digital micromirror device (24) enters the case (27) from the entrance (25) and is arranged in a large number inside the case. Reflected by a micromirror (electric mirror array). At this time, by controlling the angle of each mirror by a computer, a part of the laser light is emitted from the emission port (26) to the outside of the case (27), and the remaining laser light is emitted from the inner wall of the case (27). To absorb.

A part of the laser light emitted from the emission port (26) to the outside of the case (28) is guided to the dichroic mirror (9) disposed in the microscope (3) and is collected by the objective lens (6). To the sample cell (4).
The laser beam guided to the sample cell (4) is simultaneously focused on a plurality of locations in the vicinity of the minute object in the sample cell, thereby generating shock waves at a plurality of locations in the vicinity of the minute object. The micro object is trapped in a state of being confined by a shock wave generated from the surroundings.
Then, by moving the electric stage (5) in a state where the minute object is captured, the minute object such as a cell can be moved in the sample cell.

FIG. 6 is a diagram schematically showing a method of moving a minute object by the method as described above, and an ultrashort pulse laser (for example, femtosecond laser) (L2) output from the first laser light source (1). ) Are simultaneously condensed at a plurality of locations (indicated by black dots) in the vicinity of the periphery of the minute object (M) as indicated by arc-shaped arrows in the figure, and a shock wave is generated at each of the condensing points. Can capture a minute object. Then, by moving the electric stage (5) in this state, the minute object can be moved in the sample cell as indicated by a straight arrow.
Further, according to the apparatus of the fourth embodiment, the shock wave of the ultrashort pulse laser output from the first laser light source (1) can be simultaneously applied to a large number of minute objects. As described in the embodiment, the transfer of the minute objects in the sample cell (4) and the isolation of the cells can be simultaneously performed on a large number of minute objects.

According to the devices of the third and fourth embodiments, since there is means for capturing a micro object using the shock wave of the ultrashort pulse laser, the force of the intermediate strength between the conventional trapping laser and the mechanical manipulator It becomes possible to capture a minute object.
In the present invention, in the apparatus of the third and fourth embodiments, the second laser that outputs the trapping laser for manipulating the minute object by the radiation pressure described in the first embodiment or the second embodiment. A laser light source (2) and / or a mechanical manipulator (14) can be provided.

  Moreover, in the micro object processing apparatus according to the present invention, the sample cell (4) is composed of a plurality of micro cells connected by micro flow channels, and a sample solution containing the micro object is supplied to the sample cell (4). A configuration in which a micro liquid feeding system including a microsyringe or the like for injection / extraction is preferably employed.

Hereinafter, a minute object processing method that can be suitably implemented by using the minute object processing apparatus according to the present invention will be described by way of example.
(Example 1)
<Extraction of specific cells differentiated from stem cells>
First, as shown in FIG. 7 (a), stem cells (A) such as mesenchymal stem cells, hematopoietic stem cells and embryonic stem cells are placed on the stage (5) using a mechanical manipulator (micropipette) (14). The sample cell (substrate) (4) is injected.
The sample cell (substrate) (4) is left for about 48 hours, for example, and when the stem cell (A) is differentiated into a plurality of types of cells (B), (C), (D) (see FIG. 7B), Irradiate a specific cell (B) on the substrate (4) with an ultrashort pulse laser (femtosecond laser) (intensity 0.5 μJ / pulse, pulse width 150 femtosecond) (L2) output from the first laser light source (1) Then, the specific cells (B) are peeled from the substrate (4) (see FIG. 7C).
After detaching specific cells (B) from the substrate (4), only the detached cells (B) are collected from the substrate (4) using a mechanical manipulator (micropipette) (14) (FIG. 7 ( d)).

FIG. 8 is a schematic diagram showing the peeling operation of this specific cell (B).
Differentiated cells (B) are attached to the substrate (4) by needle-shaped limbs (filopodia) and lamellipodia (lamellipodia), and therefore cannot be moved by a laser manipulator using normal radiation pressure. When a femtosecond laser is directly irradiated to a needle-shaped temporary foot of a specific cell (B) and cut (see (a)), the cell (B) is contracted in a polka dot shape (see (b)). By irradiating a femtosecond laser (intensity 0.5 μJ / pulse, pulse width 150 femtosecond) (L2) at a position several mm away from the cell (B) (see (c)), the cell (B ) Can be peeled from the substrate (4).
The arrows in FIG. 8A indicate the operation trajectory of the femtosecond laser, and the broken line concentric circles in FIG. 8C indicate the shock wave generated by the femtosecond laser.

(Example 2)
<Isolation of specific cells from tissues and artificial insemination>
First, as shown in FIG. 9A, an ultrashort pulse laser (femtosecond laser) (intensity 1 μJ / pulse, pulse width 150 femtoseconds) output from the first laser light source (1) to the living tissue (E). ) (L2) is irradiated, and sperm cells (F) are extracted from the tissue by shock waves.
Next, egg cells (G) are extracted from the biological tissue (E) using a mechanical manipulator (micropipette) (14) (see FIG. 9B).
Then, for these extracted sperm cells (F) and egg cells (G), the sperm cells (F) are lasers produced by laser light (intensity 100 mW, continuous wave light) (L1) output from the second laser light source (2). By trapping, the egg cells (G) are arranged at positions close to each other by a mechanical manipulator (micropipette) (14) (see FIG. 9C).
Finally, an ultra-short pulse laser (femtosecond laser) output from the first laser light source (1) (intensity 0.5 μJ / pulse, pulse width) between the sperm cells (F) and egg cells (G) placed in close proximity. 150 femtoseconds (L2) is irradiated, and the sperm cells (F) and the egg cells (G) are fused by a shock wave (see FIG. 9 (d)).

  INDUSTRIAL APPLICABILITY The present invention can be widely used in various technical fields as an apparatus for processing a micro object using a laser manipulation technique, and can be suitably used as an apparatus for processing cells and the like in the field of biotechnology. .

It is the schematic which shows an example of the basic composition of the micro object processing apparatus which concerns on this invention. It is the schematic which shows the example of a change of the basic composition of the micro object processing apparatus which concerns on this invention. It is the schematic which shows another modified example (3rd embodiment) of the basic composition of the micro object processing apparatus which concerns on this invention. It is the figure which showed typically the method of moving a micro object with the apparatus of 3rd embodiment. It is the schematic which shows another modification (4th embodiment) of the basic composition of the micro object processing apparatus which concerns on this invention. It is the figure which showed typically the method of moving a micro object with the apparatus of 3rd embodiment. It is a figure which shows an example of the micro object processing method which can be implemented suitably by using the micro object processing apparatus which concerns on this invention, Comprising: The extraction method of the specific cell differentiated from the stem cell is shown. It is a schematic diagram which shows peeling operation of a specific cell. It is a figure which shows an example of the micro object processing method which can be implemented suitably by using the micro object processing apparatus which concerns on this invention, Comprising: The isolation | isolation of the specific cell from a structure | tissue and the method of artificial insemination are shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st laser light source 2 2nd laser light source 3 Microscope 4 Sample cell 5 Stage 6 Objective lens 7 Light source lamp 8 Imaging device 9 Dichroic mirror 10 Total reflection mirror 11 Electronic control mirror 12 Dichroic mirror 13 Computer 14 Mechanical manipulator 15 Laser beam separation Means 16 Electric mirror unit 24 Digital micromirror device

Claims (11)

  A first laser light source that outputs an ultrashort pulse laser for operating a minute object contained in a sample cell by a shock wave; and a second laser light source that outputs a trapping laser for operating the minute object by radiation pressure; A micro object processing apparatus comprising: a light guiding unit that guides laser beams output from the first and second laser light sources into the sample cell.   A first laser light source that outputs an ultrashort pulse laser for manipulating a minute object contained in a sample cell by a shock wave, and a mechanical manipulator for manipulating the minute object are provided. Micro object processing equipment.   2. The minute object processing apparatus according to claim 1, further comprising a mechanical manipulator for manipulating the minute object contained in the sample cell.   The micro object processing apparatus according to claim 1 or 3, wherein the light guiding means guides laser beams output from the first and second laser light sources coaxially into the sample cell.   A laser beam separating means for separating the laser beam output from the first laser light source into a plurality of beams; and a control mirror for independently controlling the separated laser beam and guiding it into the sample cell. The micro object processing apparatus according to any one of claims 1 to 4, wherein   A first laser light source that outputs an ultra-short pulse laser, and the laser light output from the first laser light source is guided to a plurality of locations near the periphery of the minute object included in the sample cell, and the minute object is captured by a shock wave. An apparatus for processing a micro object characterized by comprising a capturing means for performing the processing.   7. The minute object processing apparatus according to claim 6, wherein the capturing unit includes an electric mirror unit that scans the laser beam output from the first laser light source along the vicinity of the periphery of the minute object.   7. The micro object processing according to claim 6, wherein the capturing means comprises a digital micromirror device that simultaneously condenses the laser light output from the first laser light source at a plurality of locations in the vicinity of the periphery of the micro object. apparatus.   2. The sample cell is set on a stage of a microscope, and is configured to capture a transmission image of light irradiated from a light source of the microscope and passing through the sample cell with an imaging device. The micro object processing apparatus in any one of thru | or 8.   The micro object processing apparatus according to any one of claims 1 to 9, wherein a pulse width of the ultrashort pulse laser is several femtoseconds to several hundred picoseconds. The micro object processing apparatus according to claim 1, wherein the micro object is any one of an intracellular organelle, a cell, a living tissue, a living organ, a protein crystal, or a complex thereof.

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