JP4006519B2 - Fine particle labeling apparatus and labeling method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a labeling device and a labeling method for applying a visual label to a microparticle using laser light, and in particular, for non-contact automatic operation of minute objects and microstructures such as gene manipulation and micromachine assembly. The present invention relates to a microparticle labeling apparatus and a labeling method capable of providing a visual label that can be easily identified by image processing to microparticles that are required at the time.
[0002]
[Prior art]
By attaching a small polystyrene sphere or glass sphere to the surface of a minute substance or minute structure (hereinafter referred to as a minute object) and focusing the laser beam on these surfaces, there is no thermal damage to the minute object. It is known that a minute object can be captured and operated without contact (see Non-Patent Documents 1 and 2 below). If the attached fine particles can be visually identified, the above-mentioned non-contact manipulation of minute objects may be automated using image processing and modern control theory. Thus, for example, a fine polystyrene sphere colored with a fluorescent dye or the like is realized, and the polystyrene sphere can be used as a fine particle to be attached to a minute object to be manipulated. Various means have been developed for attaching fine particles to the surface of a minute object, such as using a photo-curing resin as an adhesive.
[0003]
[Non-Patent Document 1]
Fumito Arai, “Current Status and Issues of Nano / Micro Manipulation”, System / Control / Information, Vol.46, No.5, 2002, pp.244-251
[0004]
[Non-Patent Document 2]
Masafumi Miwa, 2 others, “Retention of microstructures by laser scanning manipulation”, Electrical Engineering E, Vol. 120, No. 7, 2000, pp.345-349
[0005]
[Problems to be solved by the invention]
However, in order to attach microparticles to the surface of a micro object for the purpose of automatic operation of the micro object, it is necessary to prepare micro particles having various kinds of labels in advance and place them at a specific place on the surface of the micro object. Since it is difficult to attach, the above-described automatic operation of minute objects without contact has not been realized yet.
[0006]
Also, when using micro polystyrene spheres colored with fluorescent dyes etc., it is difficult to take out only one micro polystyrene sphere from many micro polystyrene spheres contained in the container. It was difficult to perform the above-described attachment operation of the various kinds of fine particles without taking out the excessive fine particles that caused disturbance.
[0007]
On the other hand, if a technology is developed that provides visual labels that can be identified without using fluorescent dyes, etc., on the microparticles attached to the surface of the minute object, the microstructure will be automatically manipulated using image processing technology. Therefore, the development of a technique for imparting various visual labels to fine particles that can be stably captured by the laser manipulation technique has been one of the important issues in the above field.
[0008]
In order to solve the above-mentioned problems, the present invention may provide different visually identifiable labels on the surface of or inside a plurality of fine particles in a non-contact manner in a medium (liquid or gas). An object of the present invention is to provide a labeling apparatus and labeling method for fine particles.
[0009]
[Means for Solving the Problems]
The object of the present invention is achieved by the following means.
[0010]
That is, the particulate labeling apparatus (1) according to the present invention includes a capturing beam output section that outputs a capturing beam, a processing beam output section that outputs a processing beam, and the incident capturing beam and processing. A focus moving unit that changes and outputs an optical path of the working beam, a converging unit that focuses the capturing beam and the processing beam outputted from the focus moving unit, and a control unit; A transport beam output unit for outputting a transport beam for transporting an identification substance; And the control unit controls the focal point moving unit to capture the capturing beam in the medium including a plurality of spherical fine particles having a higher refractive index than the surrounding medium with respect to the capturing beam. Scanning a focal point on which a beam is focused by the focusing unit on a predetermined trajectory, capturing a plurality of the microparticles on the trajectory, irradiating the captured beam with the processing beam; A single hole having a predetermined size or a plurality of holes having different sizes are formed on the surface of the fine particles, and the control unit controls the focal point moving unit to change the optical path of the carrying beam, Transport the identification substance into the hole It is characterized by that.
[0011]
The particulate labeling device (2) according to the present invention includes a capture beam output unit that outputs two capture beams, a processing beam output unit that outputs a processing beam, and the incident capture unit. A focus moving unit that changes and outputs an optical path of the beam and the processing beam, a converging unit that focuses the capturing beam and the processing beam output from the focus moving unit, and a control unit; A transport beam output unit for outputting a transport beam for transporting an identification substance; In the medium including a plurality of spherical fine particles that control the focal point moving unit to be non-transparent to the capturing beam or have a refractive index lower than that of the surrounding medium. The focal points where the two capturing beams are focused by the focusing unit are scanned on a predetermined trajectory synchronously while maintaining the focal point distance wider than the diameter of the fine particles. In an area between two orbits A plurality of the fine particles Capture Irradiating the processing beam to the captured fine particles, A single hole having a predetermined size or a plurality of holes having different sizes are formed on the surface of the fine particles, and the control unit controls the focal point moving unit to change the optical path of the carrying beam, Transport the identification substance into the hole It is characterized by that.
[0012]
The particulate labeling device (3) according to the present invention comprises: A capture beam output section for outputting a capture beam, a processing beam output section for outputting a processing beam, a focus moving section for changing and outputting an optical path of the incident capture beam and the processing beam, and A focusing unit for focusing the capture beam and the processing beam output from the focus moving unit; and a control unit, wherein the control unit controls the focus moving unit and surrounds the capture beam. In the medium including a plurality of spherical fine particles having a refractive index higher than that of the medium, a focus on which the capturing beam is focused by the converging unit is scanned on a predetermined trajectory, and the plurality of fine particles are From the state where the particles are captured on the orbit and a plurality of the particles are captured, the scanning speed of the capturing beam is decreased in the vicinity of scanning at least one of the particles, and the one particle is processed. Moving the focal point of the beam, with respect to the fine particles moved to the focal point, irradiating the processing beam is subjected to a discernible processed into the fine particles It is characterized by that.
[0013]
The particulate labeling device (4) according to the present invention comprises: A capturing beam output unit that outputs two capturing beams, a processing beam output unit that outputs a processing beam, and a focus moving unit that changes and outputs an optical path of the incident capturing beam and the processing beam. A focusing unit for focusing the capturing beam and the processing beam output from the focus moving unit, and a control unit, and the control unit controls the focus moving unit to obtain the capturing beam. In the medium containing a plurality of spherical fine particles that are non-transparent or have a refractive index lower than that of the surrounding medium, the respective focal points at which the two capturing beams are focused by the focusing unit are A plurality of the fine particles are captured in a region sandwiched by two orbits by keeping the focal distance wider than the diameter of the fine particles and synchronously scanning on a predetermined orbit. Capture From this state, the scanning speed of the capturing beam is decreased in the vicinity of scanning at least one of the fine particles, one fine particle is moved to the focal point of the processing beam, and the moving beam is moved to the focal point. Irradiate the processing beam to the fine particles, and perform processing capable of distinguishing the fine particles. It is characterized by that.
[0014]
In addition, the particulate labeling device (5) according to the present invention includes the particulate labeling device ( 3 ) Or ( 4) Leave One hole having a predetermined size or a plurality of holes having different sizes are formed on the surface or inside of the fine particles by the processing. It is characterized by that.
[0015]
The particle labeling method (1) according to the present invention includes a first step of outputting a capturing beam and a plurality of spherical particles having a higher refractive index than the surrounding medium with respect to the capturing beam. In the medium, a second step of scanning the focus of the capturing beam on a predetermined trajectory to capture a plurality of the microparticles on the trajectory, and irradiating the captured microparticles with a processing beam And One hole having a predetermined size or a plurality of holes having different sizes are formed on the surface of the fine particles. The third step and A fourth step of outputting a conveying beam for conveying the identification substance; a fifth step of capturing the identification substance by the conveying beam, moving a focal position of the conveying beam, and conveying the identification substance to the hole; It is characterized by including.
[0016]
The particle labeling method (2) according to the present invention includes a first step of outputting two capture beams, and a spherical shape that is non-transparent to the capture beam or has a refractive index lower than that of the surrounding medium. In the medium containing a plurality of fine particles, the focus of each of the two capture beams is scanned on a predetermined trajectory in synchronization with the focal point being wider than the diameter of the fine particles. Let me In an area between two orbits A plurality of the fine particles Capture A second step of capturing, and a third step of irradiating a processing beam to the captured fine particles and performing an identifiable process on the fine particles; A fourth step of outputting a conveying beam for conveying the identification substance; a fifth step of capturing the identification substance by the conveying beam, moving a focal position of the conveying beam, and conveying the identification substance to the hole; It is characterized by including.
[0017]
The microparticle labeling method (3) according to the present invention comprises: A first step of outputting a capture beam; and a focus of the capture beam in a medium including a plurality of spherical particles having a higher refractive index than the surrounding medium with respect to the capture beam. A second step of scanning on the trajectory and capturing a plurality of the fine particles on the trajectory; and a third step of irradiating a processing beam to the captured fine particles and performing an identifiable processing on the fine particles. And the third step reduces the scanning speed of the capturing beam in the vicinity of scanning at least one of the particles from the state where a plurality of the particles are captured, Includes a fourth step of moving to the focus of the processing beam It is characterized by that.
[0018]
The microparticle labeling method (4) according to the present invention comprises: A first step of outputting two capture beams, and two in the medium containing a plurality of spherical particles that are non-transparent to the capture beam or have a lower refractive index than the surrounding medium. The focal point of each of the capture beams is wider than the diameter of the fine particles, and the focal point distance is maintained, and the focal point is synchronously scanned on a predetermined trajectory. A second step of capturing a plurality of the fine particles; and a third step of irradiating the captured fine particles with a processing beam and performing an identifiable process on the fine particles, and the third step includes: From the state where a plurality of the fine particles are captured, the scanning speed of the capturing beam is decreased in the vicinity of scanning at least one of the fine particles, and the single fine particle is moved to the focal point of the processing beam. Including the step It is characterized by that.
[0019]
The microparticle labeling method (5) according to the present invention comprises the above microparticle labeling method ( 3 ) Or (4 ) Leave One hole having a predetermined size or a plurality of holes having different sizes are formed on the surface or inside of the fine particles by the processing. It is characterized by that.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the accompanying drawings. That is, a microparticle labeling device and a labeling method for capturing microparticles to be labeled on a circular orbit by scanning with a laser beam and making a circular hole in the surface or inside of the captured microparticles will be described.
[0021]
FIG. 1 is a block diagram showing a schematic configuration of a microparticle labeling apparatus according to an embodiment of the present invention. The labeling device includes a control unit 5, a camera unit 4, light sources 7 and 17, and first to third focal point moving mechanisms 12 to 12 ''. The control unit 5 is a control to be described later. And a processing unit (hereinafter referred to as CPU) 20 that performs data processing, a memory unit 21 that temporarily stores data, a recording unit 22 that continuously records data, an operation unit 23, and a control interface unit 24. And a video unit 25, a data bus 26 for exchanging data within the control unit 5, and a display unit 27. The first to third focal point moving mechanisms 12 to 12 '' are provided with a control interface unit 24. Then, the laser light controlled by the CPU 20 and emitted from the light source 7 is guided to a plurality of fine particles 3 arranged in a sample surface of a microscope (not shown) to capture the fine particles 3 as described later. In FIG. 1, the path of the laser beam irradiated from the light source 7 to the fine particles 3 is omitted. The video signal from the camera unit 4 is displayed on the display unit 27 via the video unit 25, and is recorded in the recording unit 22 as digital data for each frame as necessary.
[0022]
FIG. 2 is a perspective view schematically showing a process of capturing a plurality of fine particles 3 simultaneously by scanning with a laser beam and making a hole in the surface of the captured fine particles 3. The fine particles 3 have a spherical shape, are highly transmissive to the captured laser light to be irradiated, and have a higher refractive index than the surrounding medium, such as polystyrene spheres and borosilicate glass spheres. If it is.
[0023]
The laser light emitted from the light source 7 is decomposed into two laser lights, and one of the laser lights 1 includes the plurality of fine particles 3 as described above via the first focal point moving mechanism 12. The medium is irradiated. At this time, as shown in FIG. 2, the optical path of the laser beam 1 is controlled by the first focal point moving mechanism 12 so that the focal point moves on the circular orbit 1 ″ at an appropriate constant high speed. When the finely-moving fine particles 3 are irradiated with the scanning laser beam 1 focused at a high density, the fine particles 3 are stably captured on the circular orbit 1 ″ according to the principle of optical tweezers. After capturing of the fine particles 3 is observed by the image of the camera unit 4, the processing laser light 2 is emitted from the light source 17, the optical path is changed by the third focal point moving mechanism 12 ″, and the fine particles 3 are condensed. As a result, the focal position of the processing laser beam 2 is aligned on the surface of any one of the fine particles 3 and condensed and irradiated with high density, so that the surface of or inside the captured fine particles 3 can be visually observed. At this time, the control for focusing the processing laser beam on the fine particles 3 to be drilled is obtained by processing the image picked up by the camera unit 4. This is possible by aligning the position of the processing laser beam 2 with the position of the fine particles.
[0024]
FIG. 3 is a block diagram showing in more detail an embodiment of the optical system configuration in the labeling apparatus shown in FIG. The laser light from the light source 7 is changed in diameter, that is, the spatial spread of the laser light by the beam expander 8, passes through the half-wave plate 9, and is decomposed into two-direction laser light by the first polarization beam splitter 10, 10 ′. Is done. These two laser beams pass through the first focal position moving mechanism 12 and the second focal position moving mechanism 12 ', which are configured to include a galvano mirror pair and a focal position changing lens via electromagnetic shutters 11 and 11', respectively. Then, the respective optical path axes are aligned by the second polarizing beam splitters 13 and 13 ′, and are introduced into the microscope 6 by the relay lens 14 so as to coincide with the axis of the incident illumination light.
[0025]
The two laser beams 1 and 1 ′ after decomposition are focused at the focal position by the objective lens 15 of the microscope 6 to a wavelength limit smaller than the diameter of the fine particles 3 to be labeled, for example, to a spot size of about 1 μm. Focused with high density. The control unit 5 receives the instruction from the operation unit 23, determines the trajectory 1 ", and controls the first focal position moving mechanism 12 based on the trajectory 1" and the scanning speed. The focal point can be precisely controlled so as to move on the trajectory 1 ″. Thereby, a plurality of fine particles 3 can be captured simultaneously.
[0026]
As will be described later, the other laser beam 1 ′ thus decomposed is used for the purpose of taking out the labeled microparticles from the trajectory 1 ″, and for distinguishing the microparticles 3 for the purpose of labeling the microparticles 3 ( For example, it is used for the purpose of attaching a dye reagent) to the fine particles 3.
[0027]
For the generation of the laser beam 2 for providing a label on the surface of the fine particle 3, a light source 17 that irradiates a laser beam having a wavelength that is low in permeability to the material of the fine particle 3 is used. The laser beam 2 emitted from the light source 17 is converted in diameter by the beam expander 8 'and passed through the electromagnetic shutter 11 "and then moved to the third focal position similarly to the above-described capturing laser beams 1, 1'. The two beams and the optical path axis are aligned by the beam splitter 13 ″ through the mechanism 12 ″, and introduced into the microscope 6 through the relay lens 14, and the captured fine particles 3 are punched. At this time, the focal position of the laser beam 2 for micro machining is controlled three-dimensionally by controlling the third focal position moving mechanism 12 ″ so as to be the fine particle position observed by the camera unit 4. The
[0028]
This drilling process for the fine particles 3 is performed by changing the focal position of the laser beam 2 for micro machining using the third focal position moving mechanism 12 ″, thereby moving a plurality of fine particles 3 without moving the microscope stage or the like. At the same time, it can be captured.
[0029]
Next, a microparticle labeling method using the labeling apparatus shown in FIGS. 1 and 3 will be described with reference to the flowchart shown in FIG. In the following, the CPU 20 reads data necessary for processing from the recording unit 22, performs calculation on the memory unit 21, and records the calculation result in the recording unit 22 as necessary.
[0030]
In step S1, an image including a plurality of free-moving fine particles 3 taken by the camera unit 4 is displayed on the display unit 27 via the video unit.
[0031]
In step S2, the CPU 20 provides initial parameters such as the parameters of the circular orbit 1 ″ for moving the focal point of the laser beam 1, the scanning speed (linear speed or rotational speed), and the number n of particles to be labeled, via the operation unit 23. Data input is accepted. Here, since the Z coordinate (in the direction of the optical axis of the microscope) of the circular orbit 1 ″ can be automatically determined by focusing the fine particle 3, the changeable parameter is the center of the circular orbit. Coordinates (X0, Y0, Z0) and radius r. For example, the circular orbit 1 ″ is specified by superimposing the image of the fine particle 2 displayed on the display unit 27 using the parameters (center position and radius of the circular orbit) recorded in the recording unit 22 in advance. "Is displayed, and the center and radius of the circular orbit 1" are changed by appropriately operating the operation unit 23.
[0032]
In step S3, the CPU 20 calculates a period for scanning the focal point of the laser beam 1 on the circular orbit 1 ″ based on the parameters of the circular orbit 1 ″ determined in step S2 and the scanning speed. The control parameter of the one focus moving mechanism 12 is calculated as a function of time, and the first focus moving mechanism 12 is repeatedly controlled based on these calculated functions.
[0033]
In step S4, the CPU 20 determines whether or not the fine particles 3 are stably captured on the designated circular orbit 1 ″ using the image from the camera unit 4 and is stably captured. Steps S3 and S4 are repeated until it is determined that the image is stably captured, and when it is determined that the image is stably captured, the process proceeds to Step S5, for which a known image recognition technique may be used. The video imaged by the digital signal is digitized at a predetermined time interval and recorded as a frame image, and the difference for each pixel is calculated between the frame images, and the difference value of all the pixels is less than a predetermined value, for example, the noise level It can be judged by whether or not.
[0034]
In step S5, the CPU 20 recognizes a plurality of fine particles 3 using an image from the camera unit 4, and the position coordinates (Xi, Yi, Z0) of each fine particle 3 captured on the trajectory 1 "( i = 1 to N, where N is the total number of particles 3 captured on the orbit 1 ″. Here, the Z coordinate is a constant value (Z0) as described above. For the recognition of the fine particles 3, a known image processing may be used.
[0035]
Thereafter, in step S6, the CPU 20 sets 1 to the counter j of the repetitive process, and then in step S7, the processing laser is set at a point specified by the position coordinates (Xj, Yj, Z0) determined in step S5. The third focal point moving mechanism 12 ″ is controlled so that the light 2 is condensed, and the processing laser light 2 having a predetermined pulse width is irradiated from the light source 17 j times.
[0036]
In step S8, it is determined whether or not j = n, that is, whether or not the irradiation of the processing laser beam 2 in step S7 is completed for the n fine particles 3 to be labeled. Here, it is assumed that n ≦ N, that is, in step S2, a value equal to or smaller than the number N of captured fine particles is designated as the number n to be labeled. If j = n is not determined as a result of the determination, the counter j is incremented by 1 in step S9, and then the process of step S7 is repeated. As a result of the repetition of steps S7 to S9, each of the n fine particles 3 is irradiated with the processing laser beam 2 a different number of times, and holes of different sizes that can be used as labels for identifying the respective fine particles 3 Is formed on the surface or inside.
[0037]
If it is determined that the irradiation of n particles 3 has been completed, the process proceeds to step S10, and the CPU 20 irradiates the processing laser beam 2 in step S7, and the n particles 3 are labeled. After the second focal point moving mechanism 12 'is controlled so that the laser beam 1' is focused on one of the two, the second focal point of the laser beam 1 'moves to the minute object to which the fine particles 3 are attached. The focal point moving mechanism 12 'is controlled. As a result, as shown in FIG. 5, one labeled particle 3 is captured by the laser beam 1 ′ from the plurality of particles 3 captured on the orbit 1 ″ by the laser beam 1. It can be taken out, moved to a minute object, and attached to the minute object by a predetermined method.
[0038]
In step 11, the CPU 20 determines whether or not all the n particles 3 labeled in step S <b> 7 have been taken out from the circular orbit 1 ″, and repeats the processing in step S <b> 10 until they are all taken out.
[0039]
In step S11, the CPU 20 determines whether or not there is an instruction to stop, and when determining that there is an instruction to stop, the CPU 20 determines that there has been no stop instruction by stopping the irradiation of the capturing laser beam and ending the series of processes. If so, the process returns to step S1 to repeat the series of processes described above.
[0040]
In the above description, the focus of the processing laser beam 2 is moved to make a hole in the fine particle 3. However, the circular orbit 1 ″ for scanning the capturing laser beam 1 may be moved. For example, the capturing laser The light 1 is slowly translated in a state where the circular orbit 1 ″ is scanned so that the focal point of the processing laser light 2 is positioned on the circular orbit 1 ″, and then the scanning speed of the capturing laser light 1 is reached. As a result, the whole of the plurality of fine particles 3 is rotationally moved in the scanning direction on the circular orbit 1 ″, whereby the predetermined fine particles 3 are positioned at the focal point of the processing laser beam 2. Further, only the specific fine particle 3 is scanned on the circular orbit 1 ″ by reducing only when scanning the specific fine particle 3 without reducing the scanning speed of the capturing laser beam 1 uniformly in the entire trajectory. It may be moved in the direction and positioned at the focal point of the processing laser beam 2. Here, the scanning speed of the capturing laser beam 1 for moving the microparticle 3 on the circular orbit 1 ″ is the optical characteristic of the microparticle 3. Therefore, the scanning speed of the capturing laser beam 1 is gradually increased while observing the state of the fine particles 3 from the state where the fine particles 3 are statically captured on the circular orbit 1 ″. In this case, the fine particles 3 can be processed without using the third focal position moving mechanism 12 ″.
[0041]
In addition, another laser light source for capturing microparticles and a focal point moving mechanism are provided, and the dye reagent is captured using this laser beam, and is transported and attached to the inside of the hole formed in the surface of the microparticle 3. May be. This makes it possible to apply more types of labels to the fine particles 3.
[0042]
In addition, among the two decomposed laser beams 1 and 1 ′, a laser reagent 1 ′ that is not used for generating the circular orbit 1 ″ is applied to the inside of the hole formed in the surface of the fine particle 3. It may be used for transportation.
[0043]
In the above description, the case where the size of the hole to be opened in the fine particle 3 is changed according to the number of times the laser beam for processing is irradiated is described. However, the size of the hole is changed by changing the intensity of the processing laser beam. You may let them.
[0044]
Also, a plurality of holes may be formed in one fine particle 3 for labeling. For example, if the hole size type (m type) and the presence / absence of each type of hole are combined, 2 ^m Individual identifiable codes can be applied to the fine particles 3.
[0045]
In the above description, the case where the target fine particle is transparent to the capturing laser beam and has a high refractive index has been described. However, in the case of the non-transparent or low refractive index fine particle, the above two particles are used. Are held in a cavity formed by synchronous scanning on concentric circles while maintaining the distance between the focal points of the laser beams 1 and 1 'to be larger than the diameter of the fine particles 3. Can be captured simultaneously. At this time, the two separated laser beams 1 and 1 ′ do not interfere with each other because the polarization directions are orthogonal as described above, and the intensity distribution depends on the relative positions of the two laser beams 1 and 1 ′. There is no change. Therefore, the target fine particles can be stably captured in the cavity of the light formed by the synchronous scanning of the two laser beams 1 and 1 ′.
[0046]
In the above description, the case where the capturing laser beam 1 scans on the circular orbit 1 ″ has been described. However, in order to capture a plurality of particles 3 at the same time, the scanning is not limited to a circular scan, but can be performed with an arbitrary closed curve. Furthermore, you may reciprocately scan on arbitrary curves or a straight line.
[0047]
Further, the means for capturing the fine particles 3 is not limited to the scanning of the laser beam, and can be focused with high density by a focusing means such as an optical lens. Any device that generates a capture force toward the surface may be used. For example, white light may be used, and charged particles (α particles, electrons, protons, charged elementary particles, etc.) that can be focused by an electromagnetic focusing means may be used.
[0048]
Furthermore, the present invention is not limited to the configuration of the above-described embodiment, and the design can be changed as appropriate, such as the selection of a beam for capturing fine particles and the optical system, and the order of processing can also be changed as appropriate. Is.
[0049]
【Example】
Next, the present invention will be described in more detail with reference to examples. In the labeling apparatus shown in FIGS. 1 and 3, a continuous wave Nd: YAG laser (wavelength 1064 nm, linearly polarized light) is used as a fine particle capturing laser light source, and a triple wave of a pulsed Nd: YAG laser is used as a processing laser light source ( Using a wavelength of 355 nm, an operation of making a 1 μm hole on the surface of a borosilicate glass fine particle having a diameter of about 8 μm was performed. FIG. 6 is obtained by irradiating the fine particles 18 captured by the laser beam once with a laser beam for processing once, making one hole 18 ′ on the surface, and observing the result with a CCD camera. It is In FIG. 6, by making a hole in the surface of the fine particle 18, the transmitted illumination light of the microscope is scattered at the boundary of the hole 18 ′, and the size of the hole 18 ′ can be easily observed by bright field observation of the microscope. It appears as a difference.
[0050]
Therefore, the size and number of holes opened on the surface of the fine particles can be used as a marker that can be easily discriminated by image processing such as binarization processing, and using the size and / or number of holes, By encoding, it is possible to automatically identify the fine particles.
[0051]
Since the fine particles 18 having the holes 18 'formed on the surface remain captured by the laser light even after the holes 18' are formed, the focal position of the capturing laser light is moved by the focal point moving mechanism. Thus, it can be attached to a specific structure without losing sight of the fine particles to which the cord is applied. The fine particles 19 shown in FIG. 6 are fine particles that are moved in this way and attached to the structure.
[0052]
【The invention's effect】
According to the present invention, in a medium (liquid or gas), a fine particle labeling device capable of imparting different visually identifiable labels to the surface of or inside a plurality of fine particles in a non-contact manner, and A labeling method can be provided. This makes it possible to produce as many fine particles as possible with coded labels that can be identified by bright-field microscopic observation without using a special light source for fluorescence excitation. Can move accurately to.
[0053]
In addition, it is possible to prepare fine particles with many different types of labels that can be easily identified by image processing, and attach them to specific locations on the surface of microstructures. Using this, it is possible to automatically assemble a micro structure.
[0054]
In addition, because the labeling and transporting operations of the fine particles can be performed in a non-contact manner, it is possible to work in a closed system environment that can prevent the introduction of disturbance substances and bacteria, and in a stable environment for a long time. -Automatic production of TAS and micromachines is also possible.
[0055]
In addition, the size of the hole formed in the fine particle can be easily observed as a brightness difference in a bright field state because the illumination light of the microscope is refracted or scattered at the boundary of the hole, so image processing such as binarization processing It becomes easy to automatically discriminate the code given to the microparticles using.
[Brief description of the drawings]
FIG. 1 is a block diagram showing an outline of a microparticle labeling apparatus according to an embodiment of the present invention.
FIG. 2 is a perspective view schematically showing a state in which a plurality of fine particles are captured on a scanning orbit by scanning with a laser beam.
FIG. 3 is a block diagram showing an optical system of a microparticle labeling apparatus according to an embodiment of the present invention.
FIG. 4 is a flowchart showing a microparticle labeling method according to an embodiment of the present invention.
FIG. 5 is a perspective view schematically showing a state in which a single microparticle with a label is taken out from an orbit.
FIG. 6 is a CCD camera image showing a state in which holes are made in the surface of the fine particles using the fine particle labeling apparatus according to the embodiment of the present invention.
[Explanation of symbols]
1, 1 'laser light for capturing
1 "Scanning trajectory of laser beam for capture
2 Laser beam for processing
3, 18, 19 Fine particles
3 ', 18' holes
4 Camera section
5 Control unit
6 Microscope
7 Laser light source for capture
8, 8 'beam expander
9 Half-wave version
10, 10 'first polarization beam splitter
11, 11 ', 11 "electromagnetic shutter
12, 12 ′, 12 ″ first to third focal position moving mechanism
13, 13 ', 13 "second polarizing beam splitter
14 Relay lens
15 Condensing lens
16 Sample surface
17 Laser light source for processing
20 Processing unit (CPU)
21 Memory section
22 Recording section
23 Operation unit
24 Control interface part
25 Video part
26 Bus
27 Display section

Claims (10)

A capture beam output section for outputting a capture beam;
A processing beam output section for outputting a processing beam;
A focal point moving section for changing and outputting the optical path of the incident capturing beam and processing beam; and
A focusing unit that focuses the capturing beam and the processing beam output from the focal point moving unit;
A control unit ;
A transport beam output unit for outputting a transport beam for transporting the identification substance ;
The control unit controls the focus moving unit, and in the medium including a plurality of spherical fine particles having a higher refractive index than the surrounding medium with respect to the capture beam, the capture beam is Scanning a focal point focused by the focusing unit on a predetermined trajectory, capturing a plurality of the fine particles on the trajectory;
Irradiating the processing beam to the captured fine particles, forming one hole of a predetermined size or a plurality of holes having different sizes on the surface of the fine particles,
The fine particle labeling apparatus , wherein the control unit controls the focal point moving unit to change an optical path of the carrying beam and carries the identification substance into the hole . A capture beam output section for outputting two capture beams;
A processing beam output section for outputting a processing beam;
A focal point moving section for changing and outputting the optical path of the incident capturing beam and processing beam; and
A focusing unit that focuses the capturing beam and the processing beam output from the focal point moving unit;
A control unit ;
A transport beam output unit for outputting a transport beam for transporting the identification substance ;
The control unit controls the focal point moving unit so that two particles in the medium containing a plurality of spherical fine particles that are non-transparent to the capturing beam or have a refractive index lower than that of the surrounding medium. Each of the focal points on which the capturing beam is focused by the focusing unit is scanned on a predetermined trajectory in a synchronized manner while maintaining a distance between the focal points wider than the diameter of the fine particles, and two trajectories. the region sandwiched by, and捉capturing a plurality of the fine particles,
Irradiating the processing beam to the captured fine particles, forming one hole of a predetermined size or a plurality of holes having different sizes on the surface of the fine particles,
The fine particle labeling apparatus , wherein the control unit controls the focal point moving unit to change an optical path of the carrying beam and carries the identification substance into the hole . A capture beam output section for outputting a capture beam;
A processing beam output section for outputting a processing beam;
A focal point moving section for changing and outputting the optical path of the incident capturing beam and processing beam; and
A focusing unit that focuses the capturing beam and the processing beam output from the focal point moving unit;
A control unit,
The control unit controls the focus moving unit, and in the medium including a plurality of spherical fine particles having a higher refractive index than the surrounding medium with respect to the capture beam, the capture beam is Scanning a focal point focused by the focusing unit on a predetermined trajectory, capturing a plurality of the fine particles on the trajectory;
From the state where a plurality of the fine particles are captured, the scanning speed of the capturing beam is reduced in the vicinity of scanning at least one of the fine particles, and one fine particle is focused on the processing beam. Moved to
  An apparatus for labeling microparticles, wherein the microparticles moved to the focal point are irradiated with the processing beam to perform processing that allows the microparticles to be identified. A capture beam output section for outputting two capture beams;
A processing beam output section for outputting a processing beam;
A focal point moving section for changing and outputting the optical path of the incident capturing beam and processing beam; and
A focusing unit that focuses the capturing beam and the processing beam output from the focal point moving unit;
A control unit,
The control unit controls the focal point moving unit so that two particles in the medium containing a plurality of spherical fine particles that are non-transparent to the capturing beam or have a refractive index lower than that of the surrounding medium. Each of the focal points on which the capturing beam is focused by the focusing unit is scanned on a predetermined trajectory in a synchronized manner while maintaining a distance between the focal points wider than the diameter of the fine particles, and two trajectories. Capturing a plurality of the fine particles in a region sandwiched between
From the state where a plurality of the fine particles are captured, the scanning speed of the capturing beam is decreased in the vicinity of scanning at least one of the fine particles, and the one fine particle is moved to the focal point of the processing beam,
An apparatus for labeling microparticles, wherein the microparticles moved to the focal point are irradiated with the processing beam to perform processing that allows the microparticles to be identified. The fine particle labeling device according to claim 3 or 4 , wherein one hole having a predetermined size or a plurality of holes having different sizes are formed on or in the fine particle by the processing. . A first step of outputting a capture beam;
In the medium including a plurality of spherical fine particles having a higher refractive index than the surrounding medium with respect to the capturing beam, the focus of the capturing beam is scanned on a predetermined trajectory, and the plurality of fine particles are A second step of capturing on the trajectory;
A third step of irradiating the captured fine particles with a processing beam to form one hole of a predetermined size or a plurality of holes having different sizes on the surface of the fine particles ;
A fourth step of outputting a transport beam for transporting the identification substance;
And a fifth step of capturing the identification material by the transport beam, moving a focal position of the transport beam, and transporting the identification material to the hole . A first step of outputting two capture beams;
In the medium comprising a plurality of spherical particles that are opaque to the capture beam or have a lower refractive index than the surrounding medium, the focus of each of the two capture beams is focused on the particles. holding the wide spacing of the focal point than the diameter, and by scanning synchronously on a given track, the region sandwiched by two track, a second step of捉capturing a plurality of the fine particles,
A third step of irradiating the captured fine particles with a processing beam to form one hole of a predetermined size or a plurality of holes having different sizes on the surface of the fine particles ;
A fourth step of outputting a transport beam for transporting the identification substance;
And a fifth step of capturing the identification material by the transport beam, moving a focal position of the transport beam, and transporting the identification material to the hole . A first step of outputting a capture beam;
In the medium including a plurality of spherical fine particles having a higher refractive index than the surrounding medium with respect to the capturing beam, the focus of the capturing beam is scanned on a predetermined trajectory, and the plurality of fine particles are A second step of capturing on the trajectory;
Irradiating a processing beam to the captured fine particles, and performing a process capable of distinguishing the fine particles;
From the state in which the third step captures the plurality of particles, the scanning speed of the capturing beam is decreased in the vicinity of scanning at least one particle, and one particle is removed from the processing beam. A method for labeling microparticles, comprising a fourth step of moving to a focal point. A first step of outputting two capture beams;
In the medium comprising a plurality of spherical particles that are opaque to the capture beam or have a lower refractive index than the surrounding medium, the focus of each of the two capture beams is focused on the particles. A second step of capturing a plurality of the fine particles in a region sandwiched by two orbits while maintaining a distance between the focal points wider than a diameter and synchronously scanning on a predetermined orbit;
Irradiating a processing beam to the captured fine particles, and performing a process capable of distinguishing the fine particles;
From the state in which the third step captures the plurality of particles, the scanning speed of the capturing beam is decreased in the vicinity of scanning at least one particle, and one particle is removed from the processing beam. A method for labeling microparticles, comprising a fourth step of moving to a focal point. 10. The method for labeling microparticles according to claim 8 or 9 , wherein one hole having a predetermined size or a plurality of holes having different sizes are formed on the surface or inside of the microparticles by the processing. .

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