JP2001071300A - Manipulating device and manipulating method for minute object - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manipulating device and a manipulating method for minute object allowing effective operations with optical tweezers such as picking up and moving a minute object. SOLUTION: This manipulating method for minute object picks up a minute object 3 included in liquid 2 in a container 1 using a optical pressure of light condensed by a lens 12. A target minute object 3a is found among many minute objects 3 densely existing on a bottom surface of the container 1. While the minute object 3a is placed outside (at the front of) a pick-up range T formed by condensing light, laser is irradiated to float the minute object 3a from the bottom. The target minute object 3a is placed in the pick-up range T to pick up the minute object 3a. Thus, the minute object 3a can be picked up efficiently with high workability. Since a minute object is manipulated using light outside the pick-up range of optical tweezers, degree of freedom of workability for the optical tweezers for the minute object increases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus and a method for operating a microscopic object which performs operations of capturing and moving a microscopic object such as a microorganism, animal and plant cells.

[0002]

2. Description of the Related Art In various tests and analyzes in the field of biochemistry and the like, operations of identifying, capturing, and collecting a specific minute object from minute objects such as microorganisms and animal and plant cells existing in a sample are performed. In recent years, optical tweezers have been used for capturing minute objects. The optical tweezers form an optical trap capable of capturing a fine object by the optical pressure of the collected light, and capture a fine object present in a sample solution or the like in the optical trap. . According to this capturing method, a fine object can be captured in a non-contact / non-destructive manner, and the captured fine object can be moved to an arbitrary position in a sample by scanning with light. have.

[0003]

Some of the fine objects in the sample solution exist in a dense state at the bottom of the container, but only the target fine object is captured from the dense area. It was difficult and time was spent working.

Accordingly, an object of the present invention is to provide a device and a method for operating a fine object which can efficiently capture the target fine object.

[0005]

According to a first aspect of the present invention, there is provided an apparatus for operating a fine object, comprising: a container holding portion for holding a container containing a liquid containing the fine object; a light source for emitting light; A lens that forms a capture range in which a fine object can be captured by light pressure by condensing light, a moving unit that relatively moves the capture range with respect to the container,
Control means is provided for selectively performing an operation of positioning the fine object within the capture range by controlling the moving means and an operation of positioning the fine object outside the capture range.

According to a second aspect of the present invention, there is provided a device for operating a fine object according to the first aspect of the present invention, wherein the fine object located outside the capture range is irradiated with light so that the fine object is operated. Move in the light irradiation direction.

According to a third aspect of the present invention, there is provided a method for operating a fine object using the light pressure of light condensed by a lens, wherein the fine object is captured. In the case of irradiating light in a state where the fine object is located in the capture range formed by condensing the light, when moving the fine object in the light irradiation direction, outside the capture range Light was emitted while the minute object was positioned.

According to the present invention, fine objects in a dense state can be efficiently captured with good workability. In addition, since the operation of the fine object is performed using the light that is out of the capturing range of the optical tweezers, the degree of freedom in working the fine object with the optical tweezers can be increased.

[0009]

(Embodiment 1) FIG. 1 is a cross-sectional view of a device for operating a fine object according to Embodiment 1 of the present invention, FIG. 2 is a partial cross-sectional view of the device for operating a fine object, and FIG. FIG. 4 is an explanatory diagram of a capturing range of the fine object by the operation device, FIG. 4 is a flowchart of the fine object capturing process, and FIGS. 5 and 6 are process explanatory diagrams of the fine object capturing process.

First, the structure of a device for operating a fine object will be described with reference to FIG. In FIG. 1, a liquid 2 which is a sample is stored in a transparent container 1, and a large number of microscopic objects 3 such as microorganisms and animal and plant cells are present in the liquid 2 in a floating state. These fine objects 3 are objects to be operated such as capturing and moving. The container 1 is placed and held on a holding table 4, and the holding table 4 serves as a container holding unit.

The holding table 4 is mounted on a moving stage 5. The moving stage 5 includes an X stage 5x, a Y stage 5y, and a Z stage 5z.
Driven by the YZ driver 6. By driving the moving stage 5, the liquid 2 in the container 1 moves in the X, Y, and Z directions. In addition, X used here
The YZ coordinates are fixed to the optical system described below,
The container 1 moves relatively to this coordinate system. That is, the Z coordinate Zs of the bottom surface of the container 1 changes with the movement of the Z stage 5z, and the position of the optical axis of the optical system in the X direction and the Y direction in the container 1 is determined by the X stage 5x and the Y stage 5y.
It changes with the movement.

Below the holding table 4, a laser trap unit 10 is provided. An objective lens 12 (corresponding to a lens described in each claim) is mounted on an upper part of an optical case 11 of the laser trap unit 10.
Below the mirror 2, a half mirror 13 is disposed in an oblique direction. A laser optical system including lenses 14 and 15 is provided on a side portion of the optical case 11, and a laser beam emitted from a laser irradiation unit 16 as a light source is incident on the lens 15. This laser light enters the half mirror 13 from the horizontal direction via the laser optical system, is reflected upward, and enters the objective lens 12.

When the laser beam is irradiated from the laser irradiation unit 16 in a state where the opening 4a provided in the holding table 4 is positioned with respect to the objective lens 12, the laser beam incident on the objective lens 12 is transparent. Light enters the liquid 2 through the bottom surface of the container 1. And this laser light is liquid 2
The light is condensed inside, and a trapped range (described later) by the optical trap is formed by the condensed laser light. In this capturing range, the fine object 3 can be captured in a non-contact manner by light pressure.

An image pickup lens 1 is provided below the half mirror 13.
7 and a camera 18 are provided. The camera 18
Container 1 through objective lens 12 and half mirror 13
Of a fine object in the liquid 2 of FIG. The focus of the camera 18 at the time of this imaging is set near the above-described capture range. The capturing range is fixed on an XYZ coordinate system fixed to the optical system, and the capturing position (x
(t, yt, zt).

An image processing section 19 is connected to the camera 18, and the image data obtained by the camera 18 is sent to the image processing section 19. Then, by performing image processing on the imaging data, the fine object 3 in the liquid 2 is identified,
A search process for specifying a target fine object from a plurality of fine objects is performed. By this search processing, the position of the minute object 3 to be captured in the liquid 2 is detected. The position detection result is sent to the control unit 20, and the control unit 20 controls the XYZ driver 6 based on the position detection result to drive the moving stage 5, so that the minute object 3 to be captured in the container 1 is moved. It can be positioned at a predetermined position.
In addition, by driving the moving stage 5, the container 1 can be relatively moved with respect to the capturing range formed in the liquid 2 by the laser trap unit 10. That is, the moving stage 5 is moving means for moving the capturing range relatively to the container 1.

By controlling the moving means by the control unit 20, it is possible to selectively perform the operation of positioning the fine object 3 within the capture range and the operation of positioning the fine object 3 outside the capture range. It has become. That is, the control unit 20 is control means for selectively performing an operation of positioning the fine object 3 within the capture range and an operation of positioning the fine object 3 outside the capture range.

The control unit 20 includes an input unit 21 and an output unit 2
2 are connected. The input unit 21 includes input means such as a keyboard and a mouse, and inputs operation commands and various data. The display unit 22 displays an image of the captured fine object on a screen and also displays an operation screen at the time of operation input.

Next, referring to FIGS. 2 and 3, a description will be given of the formation of the capture range of the optical trap in the liquid 2 of the container 1 by the laser trap unit 10. FIG. As shown in FIG. 2A, the laser light incident on the objective lens 12 from below is collected in the liquid
Incident inside. When the laser light is applied to the fine object 3 in the liquid 2, light pressure acts on the fine object 3. In general, a force in the direction of moving the fine object 3 in the irradiation direction (the direction in which the laser beam travels) acts on the light pressure.

At this time, the numerical aperture N
When the laser light is rapidly focused using a lens having a large A, as shown in FIG. 2B, the fine object 3 is located at a specific position in front of the focal point F (in a direction away from the objective lens 12). The region where the acting light pressures are balanced, that is, the light pressure in the irradiation direction of the laser beam, and the focal point F in the direction opposite to the irradiation direction
There is a region where the light pressure is balanced with the light, and the fine object 3 located in this region is trapped by the laser beam. Then, in this trap state, the laser light is
When scanning inside, the fine object 3 moves in the trap state. The area where this laser beam can be trapped is
It has a predetermined spread around the above-described capturing position (xt, yt, zt), and is the capturing range T of the fine object 3.

As described above, the degree of convergence of the laser beam needs to be high to form the capture range T, and the lens 12 'having a small numerical aperture NA as shown in FIG. 2C.
Is used, the capture range T is not formed because the degree of convergence of the laser light is weak. In this case, a force is applied to the fine object irradiated with the laser beam in a direction to always move in the irradiation direction.

FIG. 3 shows the behavior of the microscopic object 3 when the microscopic object 3 is located in the vicinity of the microscopic object 3 when the microscopic object 3 is formed. FIG. 3 (a)
Indicates a state in which the fine object 3 is located further forward of the capture range T (in a direction away from the objective lens 12).
When the laser light is irradiated in this state, the fine object 3 is irradiated in the irradiation direction (capture range T
(In the direction further away from the object). As a result, the fine object 3 moves in the liquid 2 in the irradiation direction of the laser beam. At this time, the container 1 is
By using together the operation of relatively approaching the object 2, the fine object 3 can be moved in the liquid 2 more efficiently as compared with the movement by laser beam irradiation alone.

FIG. 3B shows a state in which the fine object 3 is located on the near side of the capturing range T (between the capturing range T and the objective lens 12). Even in this state, a force for moving in the irradiation direction is applied to the fine object 3 by the light pressure of the laser light to be irradiated. In this case, since the capturing range T is located in the moving direction of the fine object 3, As time passes, the fine object 3 approaches the capture range T and soon moves into the capture range T and is captured. That is, if the fine object 3 is positioned in the state shown in FIG. 3B, thereafter, only the irradiation of the laser beam is continued, and the fine object 3 automatically moves to the capture range T and is captured.

In other words, even if the fine object 3 is not accurately positioned within the capture range T, the fine object 3 can be positioned within the capture preparation range R between the capture range T and the objective lens 12 shown in FIG. Once positioned, the capture of the microscopic object 3 can be performed automatically thereafter. Since the capture preparation range R is much wider than the capture range T, by utilizing this automatic capture, the positioning accuracy between the microscopic object 3 and the capture range T required when capturing by the conventional optical trap is greatly reduced. It is possible to do.

FIG. 3C shows a method of accelerating the capturing operation at the time of the automatic capturing, in which the irradiation of the laser beam is continued and the capturing range T is relatively approached to the fine object 3. Is to be moved. This allows
As compared with the case where only the fine object 3 is moved, the capture range T
The time required for the minute object 3 to reach and be captured can be reduced.

Next, the fine object capturing process by the fine object operating device will be described with reference to FIGS. 5 and 6 along the flow of FIG. Here, a process of searching for and capturing a fine object 3a to be captured from a large number of fine objects 3 that are densely arranged on the bottom surface of the container 1 is shown. FIG. 5 shows the display state of the display screen 31 of the display unit 22 in the capturing process and the positional relationship between the minute object 3 in the container 1 and the capturing range at that time. In FIG. 5, only the laser light L indicated by a solid line indicates the actual irradiation state, and the laser light L indicated by a broken line is a virtual line for illustrating the capture range T.

As shown in FIG. 5 (b), there are cases where there are a number of minute objects that the user does not want to capture near the target minute object 3a. There is a very high possibility of capturing even fine objects that you do not want to use at the same time. In such a case, the target fine object is floated from the bottom of the container 1 together with the nearby fine object, and only the target fine object 3a is captured with the distribution of the fine object sparse.

First, a search is made for a target fine object 3 with the laser irradiation turned off (ST1). This search is performed by driving the moving stage 5 to move the imaging position and recognizing and identifying the image of the fine object 3 captured by the camera 18 by the image processing unit 19. FIG. 5A shows an image 33 of the target fine object 3a from among the many fine objects 3 on the bottom surface of the container 1 as a result of the search.
Indicates a state specified in the display screen 31. In the center of the display screen 31, a mark 32 indicating a capture position (xt, yt), which is a representative point of the capture range, is displayed. Thereafter, the position coordinates (x
(0, y0) is stored in the storage device of the control unit 20 (ST
2). Instead of performing the automatic search for the target fine object 3 using the image processing, the worker visually identifies and teaches the target fine object 3 on the display screen of the display unit 22 to perform the fine object 3a. May be stored in the storage device of the control unit 20.

FIG. 5A shows a state in which the image 33 of the target minute object 3a is specified on the display screen 31 from among the many minute objects 3 on the bottom surface of the container 1 as a result of the search. . In the center of the display screen 31, a mark 32 indicating a capture position (xt, yt), which is a representative point of the capture range, is displayed. Thereafter, the position of the searched and specified minute object 3a is recognized, and the position coordinates (x0, y0, z0) of the minute object 3a are recognized.
Is stored in the storage device of the control unit 20 (ST2).

Next, as shown in FIG.
The position coordinate (x0, y0, z0) of a is the capture position (xt,
yt, zt) to move in the Z direction in the moving stage 5
Is moved (ST3). Thereby, the fine object 3 on the bottom surface in the container 1 is located in front of the capturing range T. Next, the laser irradiation by the laser irradiation unit 16 is turned ON (S
T4). As a result, the fine object 3 is irradiated with the laser light, and as shown in FIG. 5C, the fine object 3 moves upward in the liquid 2 by the light pressure of the laser light. As a result, the distribution of the fine objects in the liquid 2 becomes sparse, and a space where no fine objects exist is generated in the vicinity of the target fine object 3a.

After a predetermined time has elapsed, it is confirmed on the display screen 31 that the target fine object 3 has floated in the liquid 2 (ST5). This confirmation is performed by observing that the fine object 3 moves out of the focus position of the camera 18 due to the movement and the image 33 of the fine object is out of focus on the display screen 31.

Thereafter, the laser irradiation is turned off again (S
T6) Similar to ST1, the target fine object 3a is searched (ST7), the position of the fine object 3a is recognized, and the position coordinates (x0, y0, z0) at this time are detected. After confirming that only one fine object 3a has been searched, the moving stage 5 is moved so that the captured position (xt, yt, zt) matches the position coordinates (x0, y0, z0) of the fine object 3a. Is moved (ST8). Then, as shown in FIG. 6A, when the fine object 3a is positioned within the capture range T, the laser irradiation is turned on again to turn the fine object 3a into the capture range T as shown in FIG. 3 (ST
9). As a result, only the target fine object 3a is captured from the many fine objects existing on the bottom surface of the container 1.

The method of operating a fine object described above utilizes a characteristic that the action of the light pressure on the fine object is different between inside and outside of a capturing range T formed by condensing light with a lens. By using the optical tweezers device described above, it is possible to efficiently capture a fine object. That is, when the target fine object 3 is to be captured from among the fine objects that are densely packed at the bottom of the container, the laser light is applied to the target fine object 3a located at the bottom of the container from below (container). From the bottom) and illuminate the target fine object 3a
Then, the microscopic object 3 surrounding the target is floated in the sample solution, and when the distribution becomes sparse, the target microscopic object 3a is captured.

At this time, as the laser light used to levitate the fine object 3a, light outside the capture range (in a direction away from the objective lens) is used. Thereby, the fine object irradiated with the laser light moves in the light traveling method (irradiation direction) without being captured. Furthermore, the range of use of optical tweezers, which was conventionally used only for capturing, can be extended to effectively utilize the optical tweezers device, and the movement by light pressure of light is compared with the case of moving in the conventional capturing state. Since the moving speed can be set high, the effect of improving the operation efficiency can also be obtained.

(Embodiment 2) FIG. 7 is a sectional view of a device for operating a fine object according to Embodiment 2 of the present invention. In Embodiment 1 described above, the relative movement of the capturing range with respect to the container 1 is performed by moving the container 1 relative to the lens by the Z stage 5Z of the moving stage 5, whereas in Embodiment 2, Is performed by changing the optical distance between the capture range and the objective lens.

In FIG. 7, a holding table 4 has a moving stage 5 'composed of an X stage 5x and a Y stage 5y.
It is attached to. The holding table 4 is an XY driver 6 '
To move in the X direction and the Y direction, that is, in a plane perpendicular to the center line of the objective lens 12.
Are relatively moved in the plane.

In the above configuration, the position of the container 1 in the Z direction with respect to the objective lens 12 is fixed. Therefore, in this state, the capture range formed by being condensed by the objective lens 12 cannot be moved relative to the container 1 in the Z direction. Therefore, in the second embodiment, the capturing range is moved relative to the container 1 by the following configuration.

The lens 14 constituting the laser optical system is mounted on a horizontal moving mechanism 23. Moving mechanism 23
Is driven by the Z driver 24, the lens 14 moves in the horizontal direction, and therefore, the lens 14 moves relatively to the objective lens 12. This allows
The incident angle of the laser light incident on the objective lens 12 from below changes, and the optical distance between the capture range formed by the laser light collected by the objective lens 12 and the objective lens 12 changes. That is, the lens 14, the moving mechanism 23, and the Z driver 24 are a second moving unit that optically changes the distance between the capturing range and the objective lens 12.

By adopting such a configuration, even when the container 1 is fixed in the Z direction, the objective lens 1
The container 1 can be relatively moved with respect to the capture range formed by the light collected by the light source 2. Thereby, it becomes possible to perform the fine object capturing process similar to the example shown in the first embodiment. That is, in ST3 and ST8 of the first embodiment, instead of moving the moving stage 5, the moving mechanism 23 is driven to move the lens 14, so that the capturing position (xt, yt, zt) and the position coordinates (x0). , Y0, z0). Therefore, with the configuration shown in the second embodiment,
The same effect as in the first embodiment can be obtained.

As described above, in the device for operating a fine object according to the present invention, the light outside the capturing range of the optical tweezers is also used for operating the fine object. Manipulate fine objects (capture,
Move). In particular, a remarkable effect can be expected when a target minute object is captured from a dense minute object.

[0040]

According to the present invention, fine objects in a dense state can be efficiently captured with good workability. In addition, since the operation of the fine object is performed using the light that is out of the capturing range of the optical tweezers, the degree of freedom in working the fine object with the optical tweezers can be increased.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a fine object manipulation device according to a first embodiment of the present invention.

FIG. 2 is a partial cross-sectional view of the device for operating a fine object according to the first embodiment of the present invention;

FIG. 3 is an explanatory diagram of a capturing range of the fine object according to the first embodiment of the present invention using the operating device.

FIG. 4 is a flowchart of a fine object capturing process according to the first embodiment of the present invention.

FIG. 5 is an explanatory diagram of a process of a fine object capturing process according to the first embodiment of the present invention.

FIG. 6 is a process explanatory view of the fine object capturing process according to the first embodiment of the present invention.

FIG. 7 is a cross-sectional view of a device for operating a fine object according to the second embodiment of the present invention;

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Container 2 Liquid 3 Fine object 4 Holding table 5 Moving stage 6 XYZ driver 10 Laser trap unit 12 Objective lens 14 Lens 20 Control unit 23 Moving mechanism 24 Z driver T Capture range

Claims (3)

[Claims] 1. A container holding portion for holding a container containing a liquid containing a microscopic object, a light source for emitting light, and a fine object can be captured by light pressure by condensing light from the light source. A lens that forms a capture range, a moving unit that moves the capture range relative to the container, an operation of positioning the fine object within the capture range by controlling the moving unit, and the fine object And a control means for selectively performing an operation of positioning the object outside the capture range. 2. The apparatus for operating a fine object according to claim 1, wherein the fine object is moved in a light irradiation direction by irradiating the fine object located outside the capture range with light. 3. A method for operating a fine object using the light pressure of light condensed by a lens, the method comprising: condensing light when capturing the fine object. When irradiating light in a state where the fine object is positioned within the capture range to be formed, and when moving the fine object in the light irradiation direction, irradiating light with the fine object positioned outside the capture range A method for operating a fine object, comprising: