JP4919232B2 - Local fluorescent labeling microdevice for micro object manipulation and measurement - Google Patents

Description

  The present invention relates to a microdevice for operating a minute object. More specifically, the present invention operates a micro object in a fluid such as a liquid (which can be applied to a micro device for manipulating a micro object in a gas or a vacuum as well as in a liquid). The present invention relates to a micro device having a locally fluorescently labeled three-dimensional structure and a position / action force measuring system using the micro device.

  Conventionally, stretched glass pipettes, microtweezers manufactured using MEMS technology, microbeads, and the like have been used for manipulation and measurement of microscopic objects dispersed in a liquid. Using microspheres called microbeads captured by a laser called optical tweezers, micrometer / nanosize objects are manipulated and measured when light is incident on microspheres with a large refractive index. However, a force corresponding to the change in momentum is generated on the surface of the fine particles. In order for these resultant forces to be directed to the laser focus, the principle of working to attract fine particles is used. Above all, the technique of manipulating minute objects using microbeads captured by optical, electrical, or magnetic action force can be applied in a sealed container, and many microbeads are dispersed. It is widely used in researches dealing with cells and biomolecules because it allows manipulation of a large number of objects and has a large movable range (see Patent Document 1 below).

However, since the shape of the microbead is limited to a spherical body, it has been difficult to perform local operations such as grasping, pulling, and twisting, and single molecule operations. For this reason, various microdevices (see Non-Patent Document 1 and Patent Documents 2 and 3 below) that have a more complicated structure suitable for operation of an object and can be dispersed in a liquid like microbeads have been developed. . The present inventor has thus far developed a two-degree-of-freedom nanoneedle, a two-degree-of-freedom nanomover, and a two-degree-of-freedom nanorobot hand having a length of several μm as a light-driven nanomanipulator as described above. Has succeeded in individual remote control of live cells.
Patent Publication 2001-165840 Applied Physics Letters, Vol. 82 (1), pp. 133-135, 2003 Patent publication 2005-500914 Patent Publication 2007-97475

  Usually, these micro devices are observed in a bright field by a camera connected to a microscope. However, in research dealing with cells and biomolecules, since a target object is often stained with a fluorescent substance, a microdevice operable under fluorescence observation is required. For this purpose, it is necessary to be able to detect the position of the microdevice even under fluorescence observation. However, when the microdevice is made of a material having high fluorescence intensity, there is a problem that the fluorescence background becomes high and observation of fluorescence of the object itself is hindered.

  Therefore, the present invention solves the above-described problems and is a micro device for manipulating a minute object, which can detect a position under fluorescence observation and does not interfere with fluorescence observation of an object. The purpose is to provide a device.

For this reason, the present invention
A microdevice for enabling manipulation of a minute object, the microdevice being irradiated with a femtosecond pulse laser on a UV curable epoxy photocurable resin and three-dimensionally cured, After the uncured resin is washed, it is formed at the opposite end of the contact area with the object on the microdevice by a resin in which a fluorescent dye is mixed with a UV curable epoxy curable resin, and is captured by light pressure. And at least one part as a local region to be formed, and the one part that is three-dimensionally cured is formed as a part having a shape or a mechanism suitable for the operation of the object, and a part in contact with the object , enzymatic, protein, nucleic acid, antibody, extracellular matrix, a lipid, a sugar chain, at least one inorganic catalyst is fixed, also formed on the opposite end of the contact portion One site is made form a cylindrical projection which is caught by light pressure, a microdevice, characterized in that the projection only fluorescent labels are configured granted.
Further, the microdevice is characterized in that the dimension in any of X, Y, and Z directions of the at least one portion is less than 100 μm.
In the micro device, the dimension in the XY direction of the at least one fluorescently labeled detection region is less than 50 μm.
The microdevice is characterized in that a fluorescent label having another fluorescence characteristic is applied to the fluorescently labeled local region .
In addition, the micro device is characterized in that the plurality of fluorescently labeled local regions have different fluorescence characteristics.
The at least one fluorescently labeled local region includes a fluorescent dye, inorganic fluorescent particles, quantum dots, fluorescent beads, or a mixture thereof.
The microdevice is characterized in that the relationship between the force acting on at least one site captured in the fluid by the light pressure and the displacement from the equilibrium point is calibrated theoretically or experimentally.

  According to the present invention, it is a micro device for enabling manipulation of a minute object, and is suitable for manipulation of at least one part captured by electrostatic force, magnetic force, light pressure, fluid force, etc., and an object. And having a fluorescent label in at least one local region that is sufficiently away from the contact area with the object on the microdevice. It is possible to easily detect the position of the device and to observe the fluorescence of the object itself, and to measure the micro force and the elasticity of subcellular organelles with higher accuracy. .

In addition, when the dimension in any one of the X, Y, and Z directions of the at least one part is less than 100 μm, it is possible to more accurately operate the minute object.
In addition, when the microdevice is composed of resin, glass, metal, biopolymer, functional fine particles, or a mixture thereof, it is not affected by dissolution by the liquid and the like in the liquid. Operation becomes possible.
Furthermore, when each of the parts is formed of a different material among the materials of the resin, glass, metal, biopolymer, functional fine particles, or a mixture thereof, the appropriate material is used in the right place, The material suitable for adopting the shape suitable for the point contact, line contact, surface contact with the object to be formed, or the operation suitable for the transfer, rotation, cutting, compression, pulling, drilling, etc. of the target object can be freely selected. To improve design freedom.

Furthermore, when at least one site captured in the fluid by the electrostatic force, magnetic force, light pressure, fluid force, or the like includes at least one of dielectric fine particles, magnetic fine particles, and transparent fine particles, The force to capture the part in the fluid is improved by magnetic force, light pressure, fluid force, etc., and the object to be manipulated is more effective by a micro device including at least one of dielectric fine particles, magnetic fine particles, and transparent fine particles. Can be operated manually.
In addition, when a protein having a hydrophilic group, a polymer, or a mixture thereof is bound, mixed, adsorbed or coated on the surface of the at least one site, the adhesive strength of the surface decreases, Is easier to operate.
Furthermore, when at least one of an enzyme, protein, nucleic acid, antibody, extracellular matrix, lipid, sugar chain, and inorganic catalyst is fixed to at least one site having a shape or mechanism suitable for the operation of the object In addition, a chemical or biochemical action can be applied to the contact portion with the object, and the width of a selected area such as a test condition is increased.

Furthermore, a liquid or fine particles containing at least one of an enzyme, protein, nucleic acid, antibody, extracellular matrix, lipid, sugar chain, and inorganic catalyst in at least one site having a shape or mechanism suitable for the operation of the object. In the case where a group is supplied, a chemical or biochemical action can be continuously applied to the contact portion to the object by supplying a liquid or a fine particle group.
Furthermore, the at least one portion is formed by lithography, chemical etching, physical etching, laser removal / cutting, stereolithography, electric discharge machining, micro cutting, molding, embossing, soft lithography, imprint, inkjet, printing. When produced by either or a combination of these, processing associated with the selection of the necessity of fluorescence emission under the excitation wavelength used for fluorescence observation, particularly a fluorescent label for position detection in a local region of a microdevice Can be easily applied by various processing methods.

Further, when the dimension in the XY direction of the at least one fluorescently labeled detection region is less than 50 μm, it is possible to measure the change in elasticity or the like of a fine object on the order of μm with higher accuracy. It becomes.
In addition, when a fluorescent label having another fluorescent property is applied to a region other than the detection region labeled with the fluorescent label, not only the position of the microdevice is detected, but also the direction and orientation are accurately determined by the discriminating power of the fluorescent label. It is possible to detect well.

Furthermore, when the plurality of fluorescently labeled detection regions have different fluorescence characteristics, the plurality of detection regions can be easily identified, and the detection accuracy of the position, direction, and orientation of the microdevice is improved. .
When the at least one fluorescently labeled detection region includes any one of a fluorescent dye, an inorganic fluorescent particle, a quantum dot, a fluorescent bead, or a mixture thereof, a position detection is performed in a local region on the microdevice. As a method of applying the fluorescent label, it can be easily performed simply by fixing or encapsulating.

  Furthermore, the relationship between the force acting on at least one part captured in the fluid by the electrostatic force, magnetic force, light pressure, fluid force, etc. and the displacement from the equilibrium point is calibrated theoretically or experimentally. Can easily calculate the acting force from the object applied to the microdevice operating the object from the displacement and force calibration formula.

  As described above, according to the present invention, operations such as conveyance, rotation, cutting, compression, pulling, perforation, etc. are simultaneously applied to a target object while observing a microscopic target object with fluorescence, particularly in a liquid. The acting force from the object can be measured. As a result, a new experimental technique has been made available in which cells, biomolecules, and the like are observed at the same time while observing the fluorescence of cells and biomolecules, and the reaction to them is observed.

  Embodiments of the present invention will be described below with reference to the drawings. 1 is a schematic diagram of a local fluorescent-labeled microdevice of the present invention, FIG. 2 is a schematic diagram of an operation / measurement system using the local fluorescent-labeled microdevice of the present invention, and FIG. 3 is a cell using the microdevice and apparatus. FIG. 4 is a schematic diagram in which only the protrusions of the microdevice shown in FIG. 3 are enlarged from the upper surface, and FIG. 5 is a cell membrane obtained by compressing yeast cells obtained by the present invention. FIG. 6 is a time series diagram of the amount of displacement and the acting force on the cell membrane.

  The microdevice of the present invention is preferably composed of resin, glass, metal, biopolymer, functional fine particles, or a mixture thereof. Furthermore, a different material may be used for each part in the microdevice. At least one component of the microdevice of the present invention is made of a material that can be operated in liquid by electrostatic force, magnetic force, light pressure, fluid force, or a combination thereof, and has a shape suitable for operation. Have. The microdevice of the present invention is different from the above-described part, and at least one component is a point contact, a line contact, a surface contact with a target micro object, or a transport, rotation, cutting, compression, and tension of the target object. It has a shape suitable for operations such as drilling.

  In the part of the microdevice that comes into contact with the object, it is desirable that an appropriate polymer is present at least in a partial region on the surface for the purpose of controlling adhesiveness or non-adhesion with the object. In addition, for the purpose of adding a chemical or biochemical action to the object, at least the enzyme, protein, nucleic acid, antibody, extracellular matrix, lipid, sugar chain, inorganic catalyst is present at the contact site with the object. One may be fixed, or a liquid or fine particle group containing them may be supplied. Often, in embodiments of the present invention, the XYZ dimensions of the microdevice form a structure of less than 100 μm. More preferably, a structure in which each dimension of XYZ is less than 10 μm is formed, and more preferably, the dimension of at least one portion is less than 500 nm.

  At least one part of the microdevice of the present invention includes known lithography, chemical etching, physical etching, laser removal and cutting, stereolithography, electrical discharge machining, micro cutting, molding, embossing, soft lithography, imprint, It is produced by either ink jet printing, printing, or a combination thereof. In many cases, in the embodiments of the present invention, a material that does not emit fluorescence under the excitation wavelength used for fluorescence observation is selected as the main part of the microdevice. In the micromaterial operation using the microdevice, a fluorescent label for position detection is applied only to a local region on the microdevice that is sufficiently away from a region on the microdevice that is close to the target substance.

  As a method for imparting fluorescence to a local region on the microdevice of the present invention, a material comprising at least one of a fluorescent dye, inorganic fluorescent particles, quantum dots, and fluorescent beads is fixed to the surface of the local region, or This is realized by including in a local area. The microdevice of the present invention has at least one local fluorescently labeled detection region on the microdevice, more preferably has two fluorescently labeled detection regions, and more preferably three. It has the above-mentioned fluorescence-labeled detection region. The position of the microdevice of the present invention can be detected by optically acquiring a fluorescence signal from a detection region to which fluorescence is applied on the microdevice under fluorescence observation. When there are two fluorescent regions on the microdevice, it is possible to detect the direction as well as the position of the microdevice. Further, when there are three or more fluorescent regions, It is also possible to detect the posture.

  In many embodiments of the present invention, the microdevice is an equilibrium point with an external force due to any of electrostatic force, magnetic force, light pressure, fluid force, or a combination thereof applied for operation in liquid, That is, it has a position and orientation in which the resultant vector of external forces applied to the microdevice is zero. In such an embodiment, it is theoretically or experimentally obtained in advance from the displacement between the position and orientation of the microdevice that is operating the object using the microdevice and the position and orientation at the initial equilibrium point. From the displacement and force calibration formulas obtained, the acting force from the object applied to the microdevice that is operating the object can be obtained by calculation.

  The present embodiment is a microdevice operated by light pressure for measuring the acting force when cells are compressed in a liquid. FIG. 1 is a schematic diagram of a local fluorescently labeled microdevice used in the following examples of the present invention. In the microdevice of this embodiment, a flat plate 2 having a length of 5 μm, a height of 5 μm, and a thickness of 1 μm is fixed to the tip of a U-shaped frame 1 having a length of 5 μm, a width of 10 μm, and a height of 1 μm. A cylindrical projection 3 is formed at the end of the U-shaped frame 1 opposite to the flat plate, and fluorescence is imparted only to the projection 3. Further, the protrusion 3 is formed in an optimum shape for trapping by light pressure, and by condensing the laser light 4 through the objective lens 5 on the protrusion 3, the light pressure acts on the protrusion, and the protrusion is Captured by the laser focus 6. A wall 7 is installed at a position facing the flat plate 2, and the cell 8 placed between the flat plate 2 and the wall 7 is compressed by moving the laser focus 6.

  The above-described light-driven microdevice developed by the present inventor is produced by a two-photon nano-photofabrication method. This is a three-dimensional micromachining method using two-photon absorption, which is a nonlinear phenomenon. Two-photon absorption is a phenomenon in which two photons are absorbed simultaneously when the density of photons is very high. This phenomenon occurs only near the focal point. By utilizing this phenomenon and preparing a photo-curing resin that solidifies from a liquid to a solid by an ultraviolet laser or the like, normal one-photon absorption occurs except at the focal point, but the resin is not cured due to insufficient energy. However, since two-photon absorption occurs near the focal point, two photons are absorbed, and a phenomenon similar to that of irradiating near-infrared half-wave ultraviolet light occurs and the resin is cured. It is possible to construct an arbitrary three-dimensional macro structure by operating the laser.

  Portions other than the columnar projections 3 (1, 2, 7, hereinafter referred to as a first portion) in the present microdevice are made from a UV curable epoxy-based photocurable resin (Dimec, SCR701). This resin is three-dimensionally cured by two-photon absorption stereolithography to produce a structure. Although not illustrated in detail, first, an uncured resin is placed on a microscope slide glass, and a femtosecond pulse laser (756 nm, 80 MHz, Tsunami, Spectraphysics) collected by an objective lens is not yet used. By irradiating into the cured resin, a volume of about 100 nm square is cured. By moving the condensing point of the laser in the X, Y, and Z directions, the structure of the first portion of the target microdevice is formed. Next, after washing the uncured resin, a resin in which the fluorescent dye rhodamine B is mixed with the UV curable epoxy-based photocurable resin is placed on the slide glass on which the first portion is formed, and The structure of the cylindrical protrusion 3 is formed by the same method. Thereby, fluorescence is imparted only to the protrusions 3 of the present microdevice.

An infrared laser is used for optical driving. The light trapping force can be increased several to several tens of times by adding a cylindrical protrusion called a trap point at the rear of the light-driven microdevice. On the same principle as in the case of microbeads, the trap point is attracted to the laser focus. When the laser is scanned by the galvano scanner mirror, the micro device moves in the aqueous solution following the scanning. When the laser is focused on the trap point, the trap point is captured at the laser focus like a spring. When light is incident on a trap point with a large refractive index, the momentum of light changes due to refraction, and a force corresponding to the change in the momentum is generated on the surface of the trap point, and these resultant forces are attracted to the laser focus. Works. The greater the distance between the laser focus and the trap point, the greater the resultant force. Therefore, if the relationship between the force F acting on the microdevice and the displacement δ from the trap point center Xr to the laser focus X _L is clarified in advance, the external force acting on the trap point can be measured from the displacement δ. .

  FIG. 2 is a schematic view of an operation / measurement system using the local fluorescent labeling microdevice of the present invention used in the following examples. This apparatus system is used for operation and measurement of a microdevice. The operator operates the controller remote controller 9 and the computer 10 acquires the operation amount. The computer 10 converts the operation amount into a rotation amount of a set of galvanometer mirrors 11 and outputs the rotation amount. The infrared laser beam 12 is deflected by the galvanometer mirror 11 and is condensed through the objective lens 13 onto the projection 14 of the microdevice (corresponding to the projection 3 in FIG. 1). Since the protrusion 14 is captured at the focal point of the laser beam, the microdevice can be arbitrarily operated by operating the controller 9. The micro device 14 and the object 15 emit fluorescence by the excitation light 17 irradiated by the dichroic mirror 16 inserted in the optical path, and the fluorescence image is reflected by the dichroic mirror 18 and detected by the CCD camera 19. . The detected fluorescent image is displayed on the monitor 20 and presented to the operator, and is simultaneously acquired by the computer 10.

  FIG. 3 is a fluorescence image obtained by a CCD camera during cell manipulation using the microdevice and apparatus. Only the locally fluorescently labeled protrusion 21 (corresponding to the protrusion 3 in FIG. 1) of this microdevice has a high fluorescence signal, and the other U-shaped frame 22, the flat plate 23, the opposing wall 24 (each of FIG. 1). (Corresponding to 1, 2, 7) does not emit a fluorescent signal. In addition, the target cell 25 (corresponding to 8 in FIG. 1) does not emit fluorescence in this embodiment, but by adding a fluorescence modification corresponding to the molecule to be visualized, fluorescence from the inside or the surface of the cell is added. It is easy to observe the signal simultaneously with manipulation. Further, the position of the micro device can be accurately obtained by acquiring this CCD image in a computer and calculating the position of the center of gravity of the fluorescently labeled projection 21. Although the template matching, the luminance centroid, the edge detection, the Hough transform, and the like are used as the centroid position calculation algorithm in the present embodiment, it is not limited to these. At this time, it is possible to shorten the calculation time by previously limiting the range 26 for searching the protrusion 21 on the acquired image.

  FIG. 4 is a schematic view in which only the protrusions of the microdevice shown in FIG. 3 are enlarged from the upper surface. During the aforementioned operation, the force 26 acting on the microdevice from the cell 25 and the light pressure 27 by the laser light are in an equilibrium state. The difference 30 between the focal position 28 of the laser beam output from the computer through the galvanomirror and the barycentric position 29 of the projection calculated from the image by the CCD camera is substituted into the calibration formula 31 of the light pressure prepared in advance. Thus, the light pressure 27 acting at this time, that is, the acting force 26 from the cell is calculated.

  FIG. 5 is a time series diagram of the amount of displacement of the cell membrane and the acting force on the cell membrane at the time of compression obtained by the present invention and using yeast cells as a target example. The positional relationship between the microdevice and the cell at each time indicated by the dotted line is shown. The yeast cells are suspended in MilliQ water supplemented with 1 wt% BSA, a 5 μm opposing wall is formed in front of the microdevice, and the yeast cells are sandwiched between the opposing wall and the microdevice at a cycle of 0.5 Hz. We succeeded in measuring reaction force in real time while compressing cells. Thus, the position of the microdevice and the cell reaction force were continuously measured, and the images showed that the yeast cells were deformed by a maximum of 0.099 μm. In the present embodiment, by operating a locally fluorescently-labeled microdevice with light pressure, observation and manipulation of cells and measurement of action force without increasing the fluorescence background around the cells under fluorescence observation. Was realized at the same time.

  As described above, each embodiment of the present invention has been described. However, within the scope of the present invention, a fine object capturing mode (electrostatic force, magnetic force, light pressure, fluid force, etc.) and suitable for manipulation of an object. Device shape, mechanism, dimensions in X and Y directions, distance of fluorescent label from contact point with target object, material of micro device (resin, glass, metal, biopolymer, functional fine particle, mixture of these Or a mixture of them), characteristics of the surface material in the microdevice (a structure in which a protein having a hydrophilic group, a polymer, or a mixture thereof is bound, mixed, adsorbed or coated, etc.), the material of the capture site in the microdevice Characteristics (dielectric fine particles, magnetic fine particles, transparent fine particles, etc .. At least one of enzyme, protein, nucleic acid, antibody, extracellular matrix, lipid, sugar chain, and inorganic catalyst is solid. A liquid containing one of them, or a configuration in which a group of fine particles is supplied), shape or mechanism, and processing form of a capture site in a microdevice (lithography, chemical etching, physical etching, laser) Removal / cutting, stereolithography, electrical discharge machining, micro cutting, molding, embossing, soft lithography, imprint, ink jet, printing, or a combination thereof), fluorescently labeled detection region in the XY direction Dimensions, fluorescent properties of fluorescent labels, preparation forms of fluorescent labels in the detection area (fluorescent dyes, inorganic fluorescent particles, quantum dots, fluorescent beads, or a mixture thereof), electrostatic force, magnetic force, light pressure, Acts on at least one site that is trapped in the liquid by fluid forces, etc. And the displacement of the relationship between the equilibrium point, can be appropriately selected for theoretical or experimental calibration forms and the like. Each item in the detailed description should not be interpreted in a limited way.

It is the schematic of the local fluorescent labeling microdevice of this invention. 1 is a schematic diagram of an operation / measurement system using a local fluorescent labeling microdevice. It is the fluorescence image figure acquired with the CCD camera which is operating a cell using the said microdevice and apparatus similarly. FIG. 4 is a schematic diagram in which only the protrusions of the microdevice shown in FIG. 3 are enlarged from the upper surface. It is a time series diagram of the amount of displacement of the cell membrane and the acting force on the cell membrane at the time of compression obtained by the present invention and taking yeast cell as an example.

Explanation of symbols

1 U-shaped frame 2 Flat plate (contact part)
3 Projection for fluorescent labeling (trap point)
4 Laser light 5 Objective lens 6 Laser focus 7 Wall 8 Object (cell)

Claims (7)

A microdevice for enabling manipulation of a minute object, the microdevice being irradiated with a femtosecond pulse laser on a UV curable epoxy photocurable resin and three-dimensionally cured, After the uncured resin is washed, it is formed at the opposite end of the contact area with the object on the microdevice by a resin in which a fluorescent dye is mixed with a UV curable epoxy curable resin, and is captured by light pressure. And at least one part as a local region to be formed, and the one part that is three-dimensionally cured is formed as a part having a shape or a mechanism suitable for the operation of the object, and a part in contact with the object , enzymatic, protein, nucleic acid, antibody, extracellular matrix, a lipid, a sugar chain, at least one inorganic catalyst is fixed, also formed on the opposite end of the contact sites Microdevice one site is made form a cylindrical projection which is caught by light pressure, characterized in that the only fluorescence-labeled projection is configured granted. 2. The micro device according to claim 1, wherein a dimension of the at least one portion in any of X, Y, and Z directions is less than 100 μm . 2. The microdevice according to claim 1, wherein the dimension of the at least one fluorescently labeled detection region in the XY direction is less than 50 μm. 2. The microdevice according to claim 1, wherein a fluorescent label having another fluorescent property is applied to the fluorescently labeled local region . 2. The microdevice according to claim 1, wherein the plurality of fluorescently labeled local regions have different fluorescence characteristics. The microdevice according to claim 1, wherein the at least one fluorescently labeled local region includes any one of a fluorescent dye, inorganic fluorescent particles, quantum dots, fluorescent beads, or a mixture thereof. 2. The microdevice according to claim 1, wherein a relationship between a force acting on at least one portion captured in the fluid by the light pressure and a displacement from an equilibrium point is calibrated theoretically or experimentally. .

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