WO2005114151A1 - Measuring system - Google Patents

Abstract

A measuring system capable of appropriately evaluating response of a measuring object to a stimulus applied at a specified timing to only a part of the measuring object, e.g. a cell. An inspection system comprises an optical tweezer unit (2) for holding a fine particle (20), a lens (10) and an actuator (16) for moving the fine particle (20) toward the measuring object, a photodetector (15) divided into four and measuring a force acting from the fine particle (20) to the measuring object (35), and a confocal unit (3) and a microscope unit (4) with which the measuring object (35) can be viewed in an enlarged size.

Description

 Measurement system

 Technical field

 The present invention relates to a measurement system for measuring a response generated in a cell or the like when, for example, a cell or a biomolecule is given a stimulus by a force or a stimulus by a chemical substance. .

 Background art

 [0002] In the field of research such as cell biology, the medical field, and the pharmaceutical field, when a stimulus by a force or a chemical substance is given to a cell, a response generated in the cell is measured. The need for that is high. For example, examining the elastic properties of erythrocytes that occur when a person suffers from diabetes or examining changes in signaling pathways of cancer cells can contribute to the development of medical diagnosis and drug discovery.

 [0003] In order to observe the response of such a cell, usually, a chemical substance for stimulating the cell is sprayed near the cell with a micropipette or the like to determine how the cell responds to the chemical substance. A response is observed.

 [0004] In addition, confocal microscopes and optical tweezers described below are widely used as devices for measuring physical properties of minute measurement objects such as cells.

 [0005] A confocal microscope is a device for observing the three-dimensional shape and the spatial distribution of molecules inside a microsample of a micrometer size such as a polymer, glass, or a cell. As the name implies, a confocal microscope is characterized by the use of confocal and the use of a point light source such as a laser light source. Note that confocal refers to a state in which the light source and the photodetector are in an optically conjugated positional relationship with respect to the objective lens, that is, a state in which light emitted from one point of the light source converges on one point of the detector.

[0006] In other words, the confocal microscope focuses laser light, irradiates the object to be measured, excites fluorescence near the focus point, collects light through an optical system, and optically converges with the focus point. The light is detected through a pinhole at a conjugate position. Here, before and after the focal point, the energy density of the excitation laser light is low, so that the probability of excitation of fluorescence is reduced, and furthermore, the light passes through the pinhole. Since the probability of passing is low, so-called tomographic images can be obtained by optically cutting (optically cutting) an object having a three-dimensional structure. Furthermore, by moving the objective lens and moving the focal point forward and backward, optical cut images are acquired, and by stacking them, the three-dimensional structure of the object can be observed.

[0007] Optical tweezers have a high energy density! By condensing laser light with an objective lens and irradiating the fine particles, the radiation pressure of light is applied to the fine particles, and the fine particles are placed near the laser light spot. This device uses a phenomenon that can be caught (laser trapping). This optical tweezer can be operated by capturing particles under a microscope (Non-Patent Document 1; A. Ashkin, JMDziedic, JEBjorkholm and S. Chu, "Observation of a single-beam gradient force optical trap for dielectric particles, "Optics Letters, Vol.11, No.5, pp.288-290 (1986)), and micromanipulation.

 [0008] When the particles are caught by the optical tweezers, the particles are caught in the spot of the laser beam in a state of being hung on a panel. Here, when an external force acts on the particle, the particle is minutely displaced. By measuring the amount of displacement of the particles, the force acting on the particles can be measured.

 In order to measure the amount of mutation, scattered light is detected by irradiating particles with evanescent light, and the position of scattered light is detected by irradiating a focused beam. ing. As a typical example, an example in which the force acting between a particle and a substrate is measured with a minimum force sensitivity of the femto-Euton order (Non-Patent Document 2; "Surface-force measurement with a laser-trapped microprobe in solution" , APPLIED PHYSICS LETTERS, Vol.80 No.18, May 6 2002) and examples of measurement of the force acting between actin and myosin (Non-Patent Document 3; "Single molecule nanomanipulation of biomolecules, TRENDb in Biotechnology Vol.19 No. .6, pp 211-216, June 2001), and there are examples of measuring the force acting between DNA and RNA polymerase.

 [0010] Further, as a technique using such a confocal microscope and optical tweezers, for example, a three-dimensional image acquisition method described in Patent Document 1; Japanese Patent Application Laid-Open No. 2001-147374 (published May 29, 2001) Can be mentioned.

[0011] While applying force, a chemical substance for stimulating the cells is applied to the cells with a micropipette or the like. In the method of dissemination in the vicinity, (1) it is difficult to specify when the cells were stimulated by the stimulating substance, and (2) it is difficult to apply stimulation only to a part of the cells. There was a problem that. In addition, when stimulating cells using chemical substances, etc., it is important to evaluate (3) the power of the stimulus appropriately applied and (4) the magnitude of the applied stimulus. It was difficult.

[0012] Further, the three-dimensional image acquisition method described in Patent Document 1 only traps a small sample to be measured using optical tweezers and observes the small sample with a confocal microscope. In other words, the three-dimensional image acquisition method of Patent Document 1 considers how to apply a stimulus to a small sample fixed by optical tweezers. Therefore, the same problem as the above-described method of dispersing a chemical substance near cells using a micropipette or the like also occurs in the three-dimensional image acquisition method described in Patent Document 1.

 Disclosure of the invention

 The present invention has been made in view of the above-mentioned conventional problems, and appropriately evaluates a response of a measurement target to a stimulus given to a part of the measurement target such as a cell at a predetermined timing. It is an object of the present invention to provide a measurement system that can perform measurement.

 [0014] In order to solve the above problems, the measurement system of the present invention includes a holding means for holding the fine particles, a moving means for moving the fine particles toward the object to be measured, and a moving means from the fine particles to the object to be measured. It is characterized by comprising measuring means for measuring the amount of stimulus that acts, and observing means capable of enlarging and viewing the measurement object. Here, the “amount of stimulus” also includes the amount of a response from the object to be measured that is determined only by the amount of stimulus acting on the object to be measured. “Stimulus” and “response” include force, chemical stimulation, and thermal action.

[0015] According to the above configuration, the fine particles held by the holding means can be moved by the moving means and pressed against the measurement object. Further, if a chemical substance is fixed to the fine particles, stimulation by the chemical substance can be given to the measurement object. Therefore, if the microparticles are held at a position where the microparticles are to come into contact with the measurement target by the holding means, and the microparticles are moved to the measurement target at a predetermined timing using the moving means, a part of the measurement target can be obtained. Stimulation can be given only at a desired timing. [0016] Further, the amount of stimulus, for example, the magnitude of a force generated on the measurement target object when the fine particles are pressed against the measurement target object by the moving means can be measured by using the measurement means. Thereby, the stimulus given to the measurement object can be evaluated.

 [0017] In addition, a change in shape of the measurement object, a change in the internal structure of the measurement object, or a spatial distribution of molecules constituting the measurement object, which is caused by the fine particles being pressed against the measurement object, is as follows. It can be grasped by enlarging and observing the measurement object using observation means. Therefore, the fine particle force can also be evaluated as to whether an appropriate stimulus has been given to the measurement object.

 Further, the holding means is preferably an optical tweezer. According to this configuration, since the fine particles are held by the optical tweezers, the fine particles can be held without bringing the fine particles into contact with the holding means. Therefore, when moving the fine particles using the moving means, it is possible to prevent the holding means from applying a physical external force to the fine particles, and to prevent physical change of the fine particles such that the shape of the fine particles is deformed. Can be prevented.

 Further, it is preferable that the optical tweezers hold a plurality of fine particles by a plurality of laser beams. According to this configuration, a plurality of fine particles can be held by the optical tweezers, so that the plurality of fine particles can be pressed against the measurement target.

 [0020] For example, when a protein molecule bound to DNA is used as an object to be measured, if the fine particles are fixed to both ends of the DNA and the fine particles are fixed using optical tweezers, the relationship between the protein and DNA can be determined. Can be observed. Therefore, the measurement system of the present invention can correspond to more various measurement methods.

 [0021] Furthermore, the optical tweezers use a second lens different from the first lens used by the observation means for enlarging the object to be measured, and apply laser light to the fine particles so that the fine particles are fine. It is preferable to hold the.

 According to the above configuration, since the optical system of the optical tweezers and the optical system of the observation unit are configured independently, the holding of the fine particles by the optical tweezers and the enlargement of the measurement target by the observation unit are It can be adjusted independently. As a result, the convenience of the measurement system can be improved.

[0023] Further, the measuring means may detect that the laser light emitted from the optical tweezers unit emits the laser light. The amount of displacement of the fine particles is calculated based on the intensity of light reflected by the particles, and the above-described measurement is performed from the fine particles based on the amount of positional displacement and a panel constant indicating a holding force on the fine particles by the optical tweezers. It is preferable to measure the magnitude of the force as the amount of the stimulus acting on the object.

That is, when the fine particles are held by the optical tweezers, the holding force on the fine particles is proportional to the positional displacement amount of the fine particles. Therefore, if this proportionality coefficient is calculated in advance as a panel coefficient, the holding force can be calculated from the positional displacement amount of the fine particles and the panel coefficient.

 Therefore, in the above configuration, the position displacement amount of the fine particles is obtained using the laser light of the optical tweezers unit. That is, since the position displacement amount of the fine particles can be obtained by using the optical system of the optical tweezer unit, the configuration for calculating the position displacement amount can be simplified. Thereby, the configuration of the entire measurement system can be simplified.

 [0026] Further, the observation means is preferably a confocal microscope. That is, according to the confocal microscope, a tomographic image obtained by optically cutting the object to be measured can be obtained. Therefore, it is possible to observe changes occurring in the measurement target due to the pressing of the fine particles in more detail based on the tomographic image of the measurement target obtained using the confocal microscope.

 [0027] Furthermore, the confocal microscope has a pinhole disk having a plurality of pinholes formed on the surface thereof, and a plurality of third lenses that emit light in each of the plurality of pinholes! It is preferable that a microlens disk, a rotation unit for rotating the pinhole disk and the microlens disk, and an adjustment unit capable of adjusting a rotation speed of the disk by the rotation unit be provided.

 According to the above configuration, by rotating the pinhole disk and the microlens disk using the rotating means, the object to be measured is scanned by using the laser light passing through the third lens and the pinhole. It becomes possible.

[0029] If the rotational speed of the disk by the rotating means is set to a high speed by using the adjusting means, the object to be measured can be scanned at a high speed. This makes it possible to obtain cross-sectional images of the measurement object at short time intervals. Therefore, it is possible to observe changes occurring in the measurement object due to the pressing of the fine particles in more detail. [0030] Further, it is preferable that the observation means is for observing a change in the measurement object in a time series.

 [0031] According to the above configuration, when the measurement object changes over time due to the pressing of the fine particles, the change of the measurement object can be viewed in chronological order by the observation means. For example, when the measurement target is a cell and the microparticles have a fixed ligand that stimulates the cell, the response of the cell to the ligand can be viewed in time series.

 [0032] As described above, according to the above configuration, it is possible to observe the object to be measured which is generated by the pressing of the fine particles in more detail.

[0033] Further, the holding means, the measurement means, and the observation means may be arranged on the same side with respect to a mounting table on which the measurement target is placed.

[0034] According to the above configuration, the holding unit, the measuring unit, and the observation unit are arranged on the same side with respect to the mounting table. The space on the side can be used effectively.

[0035] Further, in the above configuration, the holding means is an optical tweezer, and the spot of the laser light of the optical tweezer is determined based on an amount of movement of the first lens used by the observation means to enlarge the object to be measured. It is preferable that a spot position changing means for changing the position is provided.

 [0036] That is, when the optical tweezers as the holding unit and the observation unit are arranged on the same side with respect to the mounting table, the laser light of the optical tweezers enters the fine particles via the first lens.

 Therefore, when the first lens moves, the spot position of the laser light of the optical tweezers is changed, and the fine particles held at the spot position also move. When the fine particles move in this way, the fine particles cannot be appropriately pressed against the object to be measured, which may be undesirable in measuring the response of the object to be measured.

Therefore, the above configuration includes a spot position changing unit that changes the spot position of the laser light of the optical tweezers based on the amount of movement of the first lens. That is, the amount of movement of the first lens and the spot position of the laser light of the optical tweezers show a predetermined relationship. This relationship If the spot position is changed using the spot position changing means based on the above, the fine particles can be held at a fixed position, and the positional relationship between the measurement object and the fine particles can be kept constant. Thereby, the fine particles can be stably pressed against the object to be measured, and the response of the object to be measured can be appropriately measured.

 [0039] Further, it is preferable that the measurement system of the present invention includes a cutting means capable of cutting the object to be measured.

[0040] According to the above configuration, by cutting the object to be measured using the cutting means, the internal structure of the object to be measured can be observed in more detail by the observation means. Therefore, it is possible to observe changes occurring inside the measurement object due to the pressing of the fine particles more minutely.

 [0041] Further, it is preferable that the cutting means irradiates the measurement target with the pulsed laser light in a focused state, and cuts the measurement target.

[0042] Still other objects, features, and advantages of the present invention will be sufficiently understood from the following description.

 Brief Description of Drawings

FIG. 1 is a diagram showing a configuration of a microscope system according to an embodiment of the present invention.

 FIG. 2 is a diagram showing a positional relationship between microparticles and cells in the microscope system of FIG. 1, together with an optical tweezer cutout and laser light from a microscope unit.

 FIG. 3 (a) is a view showing a state in which fine particles are brought close to a cell membrane.

 FIG. 3 (b) is a view showing a state in which fine particles are brought close to a cell membrane.

 FIG. 4 is a diagram showing a configuration of an analog arithmetic circuit provided in the quadrant photodetector in FIG. 1.

 FIG. 5 is a tomographic image of cells obtained using the confocal microscope of FIG. 1.

 FIG. 6 (a) is a view showing a state in which a ligand molecule is immobilized on fine particles.

 FIG. 6 (b) is a view showing a state in which a ligand molecule is immobilized on fine particles.

 FIG. 7 is a diagram showing a configuration in which a cell surgery unit is provided in the microscope system of FIG. 1.

 FIG. 8 is a diagram showing a configuration of a microscope system when a confocal microscope and an optical tweezer unit are arranged on the same side of a sample cell.

[FIG. 9] Instead of the confocal unit in the microscope system of FIG. FIG. 3 is a diagram illustrating a configuration when a focus unit is used.

 FIG. 10 is a diagram showing a configuration in which a double beam lower optical tweezer unit is used instead of the optical tweezer unit in the microscope system of FIG. 1.

 FIG. 11 is a diagram showing a state in which two microparticles are held by the microscope system in FIG. 10, and DNA elongation and interaction with DNA proteins are measured.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [1. Configuration of Microscope System]

 FIG. 1 shows a configuration of a microscope system according to an embodiment of the present invention. As shown in FIG. 1, a microscope system (measurement system) 1 according to the present embodiment includes an optical tweezer unit (holding unit) 2, a confocal unit (observation unit, confocal microscope) 3, and a microscope unit (observation microscope). Method, confocal microscope) 4. The confocal unit 3 and the microscope unit 4 constitute a confocal microscope.

 The optical tweezer unit 2 includes a lens 5, a beam splitter 6, lenses 7.8, a dichroic mirror 9, a lens (second lens, moving means) 10, a filter 11, a lens 12, a CCD camera 13, a lens 14, a four-segment photodetector (measuring means) 15, and an actuator (moving means) 16.

 With the above configuration, the optical tweezer unit 2 collimates the light of the laser light source 18 for optical tweezers guided by the optical fiber 117 by the lens 5, and then condenses the light by the lens 7. Further, the optical tweezer unit 2 reflects the light condensed by the lens 7 again using the lens 8 and then reflects the light by the dichroic mirror 9.

Then, the optical tweezer unit 2 captures the fine particles 20 in the sample cell (mounting table) 19 by condensing the light reflected by the dichroic mirror 9 with the lens 10. Further, the optical tweezer unit 2 condenses the scattered light returning from the fine particles 20 using the lens 10, reflects the power with the dichroic mirror 9, passes through the lenses 7.8 and 8 The light is reflected using the plitter 6. Thereafter, the optical tweezer unit 2 condenses the light reflected by the beam splitter 6 by a lens 14 and forms an image on a four-division photodetector 15. The four-divided light detector 15 functions to detect the position of the scattered light from the fine particles, and the detailed configuration will be described later. The confocal unit 3 includes a lens 21, a relay lens 22, Ganolevano mirrors 23 and 24, a lens 25, a two-color '14 mirror 26a, a finoletter 26b, a lens 27, and a pinhorn lens 28. I have it. The microscope unit 4 includes a lens 29, a finoletter 30, a lens (first lens) 31, and an actuator 32.

 With the above configuration, the confocal unit 3 collimates the light of the laser light source 34 guided by the optical fiber 33 using the lens 21. After that, the confocal unit 3 passes the light collimated by the lens 21 through the relay lens 22 composed of the two lenses 22a and 22b, and then reflects the light by the two galvanometer mirrors 23 and 24. The light is focused by the lens 25.

 The lens 10 of the optical tweezer unit 2 is held by an actuator 16. By operating the actuator 16, the position of the laser spot can be moved in the optical axis direction.

 Then, the microscope unit 4 collimates the light condensed by the lens 25 with the lens 29, condenses the light with the lens 31, and irradiates the measurement object 35 with the fluorescent staining. The light reflected by the measurement object 35 is collected by the lens 31 and then passes through the filter 30 so that the wavelength of the light incident from the optical tweezer unit 2 is cut off. Is reflected at

 [0052] The light reflected by the galvanomirrors 23 and 24 is reflected by the dichroic mirror 26a and passes through the filter 26b. The light that has passed through the filter 26b is collected by a lens 27 and then applied to a pinhole 28. The light passing through the pinhole 28 is detected by a photodetector 36.

 In the confocal unit 3, the light from the laser light source 34 is hardly reflected by the dichroic mirror 26a, and even if it is slightly reflected, the reflected light is cut by the filter 26b. Only the fluorescence emitted from the measurement object 35 passes through 28.

When the angles of the galvanomirrors 23 and 24 are changed, the position of the measurement target 35 where the laser light is irradiated can be changed. Therefore, the light that also returns the changed position force passes through the pinhole 28. Therefore, a two-dimensional image of the measurement target 35 can be obtained by scanning the two galvanometer mirrors 23 and 24 in synchronization with each other. [0055] Further, since the confocal unit 3 has the pinhole 28, it has confocality, and the position where the laser light is focused can be limited to one point three-dimensionally and measured. . The confocality refers to the ability to observe the object in the focal plane independently of the upper and lower structures, and the tomographic image of the object can be observed with a confocal microscope. Is also a force that has Therefore, the two-dimensional image obtained by the confocal microscope becomes a tomographic image of the measurement object.

 The lens 31 of the microscope unit 4 is held by an actuator 32. Therefore, by operating the actuator 32, the lens 31 can be moved in the optical axis direction of the laser light. Further, in synchronization with the scanning of the galvanometer mirrors 23 and 24 to obtain a tomographic image, the actuator 32 is operated to move the lens 31 in the optical axis direction, so that a plurality of tomographic images of the measurement object 35 are obtained. An image can be obtained. Thereby, the three-dimensional substance distribution inside the measurement target 35 can be measured.

 The optical tweezer unit 2 is provided with a CCD camera 13. Through the CCD camera 13, an enlarged image of the measurement object 35 can be observed in time series. Further, the optical positional relationship between the optical tweezer unit 2, the confocal unit 3, and the microscope unit 4 can be adjusted using the CCD camera 13.

 That is, while the optical tweezer unit 2 is arranged above the sample cell 19, the confocal unit 3 and the microscope unit 4 are arranged below the sample cell 19, and The unit 2, the confocal unit 3 and the microscope unit 4 are in an optically independent relationship. Therefore, it is necessary to determine the position of the optical system of each unit at the beginning of the experiment. For that purpose, the confocal laser light from the microscope unit 4 is checked using the CCD camera 13, and the optical system may be adjusted using the laser light as a mark.

[0059] Further, by installing the optical tweezer unit 2 above the sample cell 19, when the microparticles 20 captured by the optical tweezers are brought into contact with the cells as the measurement target 35, the microparticles 20 and the cells are dissociated. The focal position on the observation side can be changed without changing the positional relationship. In other words, when taking a three-dimensional image of cells with a confocal microscope, the force that moves the lens 31 on the observation side in the direction of the optical axis to change the focal plane and obtain two-dimensional images sequentially. Like Even if the lens 31 is moved, the optical positional relationship of each component in the optical tweezer unit 2 is not affected at all. Therefore, since the position of the fine particles 20 captured by the optical tweezers does not change, a three-dimensional image of the cell can be obtained by changing the focal plane.

 FIG. 2 shows a state in which the microparticles 20 are brought close to the cells as the measurement object 35 and the cells are observed. As shown in FIG. 2, the laser light from the optical tweezer unit 2 is incident on the sample cell 19. The fine particles 20 are captured by the laser light.

 [0061] Here, when the microparticles 20 are brought closer to the cells, stimulation or force is applied to the cells, and the microparticles 20 are subjected to the force generated by the interaction with molecules on the cell surface or the cell membrane. The force that is pushed back works.

 [0062] In addition, cells stimulated by the microparticles 20 exhibit a response (such as generation of calcium waves) to the cells or undergo elastic deformation. In this embodiment, the response of the cells to the stimulus, the elastic deformation, and the like are observed with the confocal microscope described above.

 [0063] Further, the position of the fine particles 20 is displaced by a force that also applies a cellular force to the fine particles 20. Here, the state of capturing fine particles with optical tweezers is similar to the state of hanging fine particles on a panel, so it is important to understand that when an external force acts on the fine particles, the length of the panel changes. Can be. If the panel constant of the optical tweezers is obtained in advance, the force acting on the particles can be obtained by measuring the amount of displacement of the particles. The procedure for obtaining the force acting on the fine particles will be described later.

 FIGS. 3 (a) and 3 (b) show a state where the microparticles 20 are brought close to the cell membrane. When the microparticles 20 captured by the optical tweezers are brought close to the cell membrane, a mutual interaction between the cell membrane and the microparticles 20 is generated. Further, when a substance serving as a ligand is fixed on the surface of the fine particles 20, the receptor molecules on the cell membrane and the fine particles 20 interact with each other. The interaction forces generated at this time are electrostatic force, dipole interaction, and van der Waals force.

 [2. Procedure for calculating interaction force]

As shown in FIG. 4, the quadrant photodetector 15 has its detection area equally divided into four fan-shaped areas A, B, C, and D. In these four sector areas A, B, C, and D, Is formed and the light intensity is detected. Then, the light intensity is subjected to arithmetic processing by an analog arithmetic circuit (measuring means) 37, and then output to a computer.

 The analog arithmetic circuit 37 is composed of three subtractors 38... And two calo calculators 39. The analog operation circuit 37 has three output terminals. Let the levels of the signals output from each of these three output terminals be I, I, and I, and let the levels of the signals detected in each of the areas A, B, c, and D of the quadrant detector be I, Ι, Ι , Ι, I, I

 A B C D

 , I are set as follows:

 z

 [0067] I = (1 +1)-(1 +1)

 A B C D

 I = (I + I)-(I +1)

 A c B D

 I = 1 +1 +1 +1

 z A B C D

 Then, in the microscope system of the present embodiment, the displacement of the fine particles can be calculated as follows using these signal levels I 1, I 2, and I 3.

 First, displacements in the X direction and the y direction, which are directions perpendicular to the optical axis of the laser light of the optical tweezers, will be described. That is, the displacement of the particles at the equilibrium position force is sufficiently smaller than the spot diameter of the laser beam! / In this case, if the displacements of the particles in the X and y directions are d and d, respectively, The relations d = al and d = bi hold between the signal levels I and I, where a, b and c are proportional constants. Using this relationship, the force acting on the fine particles is determined. The “equilibrium position” is a position of the fine particles when the fine particles are stably trapped by the optical tweezers.

 First, assuming that the panel constants of the trapping forces acting in the X and y directions are k and k, considering the property of the particles moving in Brownian motion, these panel constants are calculated using d and d as follows. Can be expressed as follows. That is, the values obtained by time-averaging the amount of deviation of the values of I and I at each time with respect to the average value of these values when I and I are measured for a predetermined time are <I> and < I>. Further, if the time average of the displacement amount of the fine particles is d>, <d>, then <d> = a <I>, <d> = b <I> can be obtained. Furthermore, between <d>, <d> and k, k

, The following relationship holds.

[0071] 1 / 2KT = l / 2k <d> ² = l / 2k <d Ϋ where K is Boltzmann's constant and Τ is absolute temperature Degrees.

 Based on the above equation, the following equation can be derived.

[0073] k = KT / (a ² <I> ² )

k = KT / (b ² <I> ² )

 [0074] Then, the force F acting on the fine particles is expressed as F = kd, and the potential U is expressed as U = Fd (i is any of X, y, z).

F = {KT / (a ² <I> ² )}-aI = {KT / (a <I> ² )} 1

U = FaI = {KT / (<I> ² ) ll ²

F = {KT / (b ² <I> ² )}-bI = {KT / (b <I> ² )} l

u = {FbI = KT / (<I> ² )} I ²

 When obtaining the forces F 1 and F 2 from these equations, it is necessary to obtain the proportional coefficients a and b.

It can be seen that the potential can be obtained even if these proportional coefficients are unknown.

 [0077] In order to obtain the proportional coefficients a and b, the sample stage is moved at a constant velocity V, and the moving distance force Panel constant k at that time is calculated. k and k. In other words, the radius of the particle ¾: The viscosity of the surrounding medium is r ?, and the displacement of the particle when the sample stage is moved in the X direction at a constant velocity V with the particle trapped is d = al. Then, the force F acting on the fine particles by the law of Stotas is

 F = k d = k al = D π η rv

 It is expressed.

 Then, k = 6π 7? Rv / (al). When k obtained in this way is compared with k obtained from Brownian motion,

k = KT / (a ² <I> ² ) = 6π ηνν / (al)

 So a is

a = KTI Ζ (6π rjrv <I> ² )

 Like ι,

b = KTI / (6π rjrv <I> ² ).

 [0079] Using these proportional coefficients, the measured force of I and I can be used to determine the actual position displacement.

. Further, the force acting on the fine particles can be obtained. Next, the displacement in the optical axis direction will be described. When a particle moves in the z direction, the relationship between the amount of movement d of the particle and the intensity of scattered light I from the particle is approximately represented by a quadratic function if the position displacement of the particle is small. Then, when the relationship between the scattered light intensity I and the position displacement d is approximated by a quadratic function,

 And I force d can be obtained.

 [0081] From this equation

d = (lp ² -qp ² ) ^1/2 = p (l -q) ^1/2

 It is expressed.

 [0082] Therefore,

^{<D> 2 = p 2 <} (I q) 1/2> 2 , and the

k = KT / (p ² <(l -q) ^1/2 > ² )

 Is given as a panel constant k in the Z direction.

 [0083] Therefore,

F = {KT / (p ² <(l -q) ^1/2 > ² )} p (l -q) ^1/2

= {KT / (p <(l -q) ^1/2 > ² )} (l -q) ^1/2

 z z

U z = {KTZ (P <(I z -q) ^1/2 > ² )} (lz -q) ^1/2 p (lz

= {KT / (<(l -q) ^1/2 > ² )} (l -q)

 z z

 It becomes.

 [0084] The coefficients p and q must be determined by experiments. To determine the coefficients p and q, repeat the measurement of the panel constant k by changing the laser light intensity of the optical tweezers, and take advantage of the fact that the panel constant changes in proportion to the light intensity. P and q can be determined by the least-squares method to satisfy. In this case, the intensity of the scattered light from the fine particles is proportional to the intensity of one light beam of the laser.

[0085] Regarding the movement of the fine particles in the z direction, generally, with optical tweezers, the fine particles are stably caught on the downstream side of the laser beam from the spot. Since the intensity of scattered light I from the particle is maximum when the center of the particle coincides with the focal point in the spot, the particle Being downstream from the pot means that I is slightly higher than the maximum, vj. This stable state force I increases when the particles move toward the light source, and decreases when the particles move downstream of the laser beam.

 [0086] Further, it can be assumed that the displacement amount of the fine particles measured by the microscope system 1 is smaller than the distance between the center of the fine particles and the focal point in the spot. Therefore, I

 Uncertainty in the direction of movement of the particles near the maximum z is not particularly a problem in measuring the force exerted on the cells by the particles! /.

 In the measurement system of the position of the fine particles by the four-divided photodetector 15 of the present embodiment, the fine particles are irradiated with a laser beam (for example, a guide light) different from the laser for optical tweezers to scatter the fine particles. It can also be realized by detecting light in a similar manner.

 [0088] Specifically, a He—Ne laser (wavelength 632.8 nm) is incident on fine particles coaxially with an Nd: YAG laser (wavelength 1064 nm) for optical tweezers, and the light of the He Ne laser scattered by the fine particles. It was confirmed that the position displacement of the fine particles could be detected by detecting the particles with the four-segment photodetector 15.

 As described above, by using a laser different from the laser for optical tweezers to measure the displacement of the fine particles, the sensitivity of the four-segment photodetector 15 can be easily adjusted. Whether the laser for optical tweezers or the other laser is detected by the quadrant photodetector 15 should be determined depending on the object to be observed and the observation method.

 [0090] Further, even if a position "sensitive" detector (PSD) is used as a device for detecting scattered light from the fine particles, the positional deviation of the particles can be similarly detected. The position sensitive detector is a device that outputs the spot position on the entire surface of the detector as a voltage with each value of the X coordinate and the y coordinate. By disposing this detector in place of the four-segment photodetector 15 after the lens 14 (see Fig. 1), it is possible to detect the position of the particles by projecting the scattered light of even fine particles onto the entire detector. .

 [3. Contact Experiment with Cell]

A contact experiment with cells was performed using the microscope system of the present embodiment, and the results will be described below. In addition, an Nd: YAG laser having a wavelength of 1064 nm was used as a laser for optical tweezers, and fine particles having a diameter of 1 μm were used. And fine particles The particles were caught by optical tweezers, and the microparticles were brought into contact with the cells by gradually moving the lens 10 (see Fig. 1) in the optical axis direction.

 [0092] Then, it was confirmed that when the microparticles were brought into contact with the cells, the pattern of the laser light that was scattered by the microparticles was changed, and the microparticles were displaced. It was also confirmed that when the microparticles were moved sideways while in contact with the cells, the microparticles shifted laterally, causing the center of the scattered light to shift laterally. These pattern changes and lateral displacement of the center of the scattered light were detected by the four-divided photodetector 15.

 [0093] FIG. 5 shows a tomographic image of cells obtained by using a confocal microscope. The cells are human vascular endothelial cells and are fixed in a state of being spread on the cover glass. The actin fibers in the cells were stained using Alexa488-Faroidine (Molecular Probes Inc.) and observed with a confocal microscope. As shown in FIG. 5, actin fibers can be observed inside the cells. Particles caught by optical tweezers cannot be observed in the tomographic image in Fig. 5.

 [4. Experiment on Stimulation with Chemical Substances]

 In addition, a method for immobilizing a molecule (ligand) as a stimulator on the surface of microparticles in order to stimulate a cell with a chemical substance and stimulating a receptor that interacts with the molecule is described below. . In order to immobilize the ligand on the surface of the microparticles, for example, a method using a crosslinker molecule, a method in which a biotin molecule attached to the ligand is bound to streptavidin molecules immobilized on the surface of the microparticles, a CyBr method, a silane coating method, or the like. You can use it!

 FIG. 6 shows the state of interaction between the microparticles and the receptor in each of these methods. First, in the method using one crosslinker molecule, as shown in FIG. 6 (a), one crosslinker molecule 40 is fixed on the surface of the fine particles 20, and a ligand molecule 41 is further fixed on one crosslinker molecule 40. In this state, when the ligand molecule 41 is brought close to the lipid bilayer (measurement target) 42 of the cell membrane, the ligand molecule 41 can bind to the receptor molecule (measurement target) 43 existing therein.

 [0096] The crosslinker molecule 40 was used as DSP (Pierce, Double-AgentTM

Cross-linker Number: 22585) A molecule or the like can be used. DSP molecules are: As shown in the chemical formula, it is a molecule having terminals at both ends that are reactive with a thiol group and an amino group. With the above configuration, the DSP molecule can react with the thiol group fixed on the surface of the fine particle 20 and the amino group contained in the ligand molecule 41 to fix the ligand molecule 41 on the surface of the fine particle 20.

[0097] [Formula 1]

Chemical formula of DSP molecule

[0098] Next, a fixing method using biotin will be described. As shown in FIG. 6 (b), a biotin molecule 44 is immobilized on the surface of the microparticle 20, and a streptavidin molecule 45 is bound thereto. Since the streptavidin molecule 45 has four binding sites, the ligand molecule 41 is immobilized on the opposite binding site.

[0099] When the microparticles 20 having the ligand molecules 41 immobilized on the surface in this manner are brought close to the cell surface and stimulated, the stimulation by the ligand molecules 41 is performed only in the area having a smaller sectional area force than the microparticles 20. Is given. For example, if the cell surface is stimulated by immobilizing the ligand molecule 41 on the surface of a microparticle 20 having a diameter of: L m, if the cell surface is recessed by 0.1 μm due to contact of the microparticle, then Is 2.8 μm ² .

 [5. Application to Cell Surgery Unit]

As shown in FIG. 7, the microscope system 1 having the above-described configuration can add a cell operation unit (cutting means) 50. This cell surgery unit 50 irradiates a cell with a femtosecond pulsed laser such as a titanium sapphire laser into the microscope optical system and focuses the laser to cut the cell membrane and organelles. Is what you do. Hereinafter, the configuration of the cell surgery unit 50 will be described. Members having the same functions as those of the microscope system 1 shown in FIG. 1 are denoted by the same reference numerals in FIG. 7, and detailed description thereof will be omitted. [0101] The cell operation unit 50 includes a laser light source 51, a galvanomirror unit 52, a shirt 53, a lens 54, and an actuator 55.

 [0102] The galvanomirror unit 52 is used to move the spot of the laser light emitted from the laser light source 51, and is used to join two galvanomirrors (not shown) at a right angle. Be composed. Furthermore, the galvanomirror unit 52 includes a shutter 53 for turning on / off the laser emitted from the laser light source 51.

 [0103] The laser light emitted from the laser light source 51 for cell cutting is reflected by the galvanomirror unit 52 and then collected by the lens 54. The lens 54 is provided with an actuator 55. By operating the actuator 55, the lens 54 can be moved and the height of the laser spot in the sample cell 19 can be changed.

 Further, the laser light condensed by the lens 54 is collimated by the lens 56 in the microscope unit 4 and then reflected by the dichroic mirror 57 before being incident on the lens 31. In this manner, the laser light emitted from the laser light source 51 can be focused on the cells in the sample cell 19. The laser light of the laser light source 51 reflected by the cells is guided to the confocal unit 3 after being absorbed by the filter 58.

 When the cell membrane is cut using the cell surgery unit 50 having the above configuration, the following measurement can be realized. First, a lateral force is applied to the cells by pressing the microparticles 20 trapped by the optical tweezers against the cells as the measurement object 35 or by trapping the microparticles 20 fixed to the cells by the optical tweezers.

 [0106] Next, the cell membrane near the place where the force is applied to the cell is cut using the cell surgery unit 50, and the movement of the fine particles 20 at that time is measured, and the morphological change of the cell is measured. Observe with a confocal microscope. As a result, the physical strength of the cell membrane and the cell structure such as the skeleton inside the cell can be known.

[0107] As the laser of the cell surgery unit 50, a titanium sapphire laser (tunable at a wavelength of 760 to 900 nm, pulse width of 80 fs, and maximum output of 800 mW) can be used. The present inventors have confirmed that a cell membrane can be cut when laser light is incident at a wavelength of 800 nm. In addition, the present inventors considered that the fine particles 20 fixed to the cell membrane surface at this time Then, it was confirmed that when a force was applied in the lateral direction of the cell membrane using the optical tweezer unit 2, the microparticles 20 were displaced when the cell membrane was cut.

 [6. Other Examples of Arrangement of Optical Tweezers]

 Further, in the microscope system 1 of the present embodiment, as shown in FIG. 8, a confocal microscope for the lower side of the sample cell 19, that is, the sample cell 19, is used instead of the optical tweezer unit 2 (see FIG. 1). It is possible to provide another optical tweezer unit (hereinafter simply referred to as lower optical tweezer unit 60) on the same side as the side on which it is arranged. Hereinafter, the configuration of the lower optical tweezer unit (holding means) 60 will be described. Note that members having the same functions as those of the microscope system 1 shown in FIGS. 1 and 7 are denoted by the same reference numerals in FIG. 8, and detailed description thereof will be omitted.

 As shown in FIG. 8, the lower optical tweezer unit 60 includes a laser light source 61, a mirror 62, a galvano mirror unit 63, a lens (moving means, spot position changing means) 64, and an actuator. (Moving means, spot position changing means) 65, a beam splitter 66, a lens 67, and a four-divided photodetector 15.

 [0110] The light emitted from the laser light source 61 passes through the beam splitter 66, is reflected by the galvano mirror unit 63, and is condensed by the lens 64. The laser light condensed by the lens 64 in this way is collimated by the lens 68 in the microscope unit 4 and reflected by the two dichroic mirrors 57 and 69 before being incident on the lens 31.

 The laser light of the laser light source 61 incident on the lens 31 in this manner is scattered by the fine particles 20. Then, after the scattered light is collected by the lens 31, it is reflected by the dichroic mirrors 57 and 69, and then passes through the two lenses 64 and 68. Further, the light that has passed through the two lenses 64 and 68 is reflected by the galvanomirror unit 63 and the beam splitter 66 and then collected by the lens 67. The light condensed by the lens 67 as described above is detected by the four-divided photodetector 15.

The lower optical tweezer unit 60 includes an actuator 65 for changing a distance between the lens 64 and the lens 68. The actuator 65 is provided on the lens 64. When the actuator 65 is operated, the lens 64 moves. When the distance between the lens 64 and the lens 68 is changed in this way, the laser The height of the microparticle 20 can be moved in the optical axis direction, and the height of the fine particles 20 trapped by the optical tweezers can be changed.

[0113] By using the mechanism for changing the height of the fine particles 20 described above, a three-dimensional tomographic image of the measurement object can be obtained with a confocal microscope without changing the positional relationship between the measurement object 35 and the fine particles 20. You can get it.

 [0114] That is, when tomographic images at various heights of the measurement object are captured using the confocal microscope, the lens 31 is moved in the optical axis direction. By operating the actuator 65 in synchronization with the movement of the lens 31 as described later, the height of the laser spot emitted from the laser light source 61 can be moved in synchronization with the movement of the lens 31. it can. This makes it possible to keep the positional relationship between the measurement object 35 and the fine particles 20 constant.

 [0115] A method of changing the distance between the lens 64 and the lens 68 in synchronization with the movement of the lens 31 will be described. First, when the distance between the lens 64 and the lens 68 is changed by d, and the distance that the fine particles 20 move in the optical axis direction is g, the relationship g = Ad holds. A is the longitudinal magnification of the optical system composed of the lens 68 and the lens 31.

 On the other hand, when the lens 31 moves by the distance P in the optical axis direction, the distance by which the fine particles 20 move in the optical axis direction in the sample cell 19 is h, and the refraction of the measurement object in the sample cell 19 is h. Assuming that the index is n and the index between the lens 31 and the object to be measured is n, the refractive index of the matching liquid (matching oil, water or air) is represented as h = P (n Zn).

 0 0 1

 [0117] g and h in the above equation are common as the spot position of one laser of the optical tweezers for trapping the fine particles 20. Therefore, in order not to change the positional relationship between the measurement target 35 and the fine particles 20, the distance d is set so that the relationship of Ad = P (n Zn) is satisfied.

 0 1

 You only need to change it.

In order to move the spot height of the lower optical tweezers unit 60 in synchronization with the movement of the lens 31 in the present embodiment, the wavefront of the light incident on the lens 31 may be curved. Therefore, even if a deformable mirror that bends the reflecting surface slightly to bend the wavefront of the reflected light or a phase shift device that can locally change the phase of the diffracted light is used, the measurement target 35 is not affected. Measured with a confocal microscope without changing the positional relationship with the fine particles 20 3D tomographic images of elephants can be obtained.

 The characteristics of the dichroic mirrors 57 and 69 and the filter 58 used in the microscope system 1 having the configuration shown in FIG. 8 will be described. The dichroic mirror 69 reflects the light of the laser light source 61 provided in the lower optical tweezers unit 60, while transmitting the light of the laser light source 51 provided in the cell operation unit 50.

 [0120] The dichroic mirror 57 reflects the laser emitted from the laser light source 51 and the laser light source 61, and emits the sample power with the laser emitted from the laser light source 34 of the confocal microscope. It transmits fluorescent light.

 [0121] Specifically, the laser light source 61 of the lower optical tweezer unit 60 is a Nd: YAG laser light source having a wavelength of 1064 nm, the laser light source 51 of the cell surgery unit 50 is a titanium sapphire laser light source having a wavelength of 760 to 900 nm, and confocal. When an argon laser light source with a wavelength of 488 nm is used as the laser light source 34 of the microscope and the wavelength range of the fluorescence excited by the argon laser is 500 to 650 nm, the wavelength characteristics of the dichroic mirrors 57 and 69 are as follows. Set it to.

 First, the dichroic mirror 69 may be a mirror that reflects a wavelength longer than 950 nm and transmits a wavelength shorter than 950 nm. Further, the dichroic mirror of the dichroic mirror 57 may be a mirror that reflects a wavelength longer than 700 nm and transmits a wavelength shorter than 700 nm. The filter 58 may be a filter that absorbs a wavelength longer than 700 nm and transmits a shorter wavelength.

 [7. Other Configuration Examples of Confocal Microscope]

 In the microscope system 1 described above, the configuration using the units using the Ganolevano mirrors 23 and 24 as the confocal unit 3 has been described (see FIG. 1), but the configuration of the confocal unit 3 is not limited to this. .

For example, as shown in FIG. 9, instead of the confocal unit 3, a multi-pinhole confocal unit (observation means, confocal microscope) 70 can be used. As the multi-pinhole confocal unit 70, for example, a unit commercially available from Yokogawa Electric Corporation can be used. Hereinafter, the configuration of the multi-pinhole confocal unit 70 will be described. Note that members having the same functions as those of the microscope system 1 described above are the same in FIG. The detailed description is omitted by giving the same reference numeral.

 As shown in FIG. 9, the multi-pinhole confocal unit 70 includes lenses 21 and 27, a micro lens disk 71, a pinhole disk 72, and a motor for rotating these disks 71 and 72. (Rotating means) 73, a dichroic mirror 74, and adjusting means (not shown) for adjusting the rotation speed of the motor 73.

 [0126] The microlens disc 71 includes a plurality of lenses (third lens, not shown) for condensing and irradiating light to each of the pinholes formed on the surface of the pinhole disc 72. Is what it is. The pinhole disk 72 has a large number of pinholes (not shown) formed on the surface thereof.

 With the above configuration, the multi-pinhole confocal unit 70 reflects the laser beam returning from the sample cell 19 and passing through the pinhole disk 72 by the dichroic mirror 74. The laser light reflected by the dichroic mirror 74 is condensed by the lens 27 and detected by the CCD camera (observing means) 75.

 When the multi-pinhole confocal unit 70 is used, a tomographic image of the object to be measured can be obtained using the CCD camera 75 without scanning by a galvanomirror. In addition, approximately 1000 points in the object to be measured can be excited simultaneously to detect light emission, making it suitable for high-speed image capture!

 When the multi-pinhole confocal unit 70 is used, more than 500 tomographic images of the object to be measured can be captured per second, and when the lens 31 is moved in synchronization with the capturing of the image, 30 tons per second can be obtained. 3D images can be captured faster than the set. Therefore, when observing the measurement object in time series, a tomographic image of the measurement object can be obtained at shorter time intervals, and the time resolution of the measurement result can be significantly improved.

 [8. Other Configuration Examples of Optical Tweezers]

 In the above description, the case where the optical tweezer unit 2 (see FIG. 1 and the like) and the lower optical tweezer unit 60 (see FIG. 8) are employed as the configuration for trapping the fine particles 20 has been described. It is also possible to employ a double beam lower optical tweezers cut (holding means) 80 described below with reference to FIG.

[0131] The double beam lower optical tweezer unit 80 is a two-beam laser for optical tweezers. A laser can enter and hold two particles at the same time. More specifically, as shown in FIG. 10, the double beam lower optical tweezer unit 80 includes a laser light source 81, a polarizing beam splitter 82a '82b, a ganole mirror unit 83a' 83b, and a lens 84 (movable Means, spot position changing means), actuator (moving means, spot position changing means) 85, half-wave plates 86a, 86b, 89, and four-segment photodetector (measuring means) 90a-90b. ing.

 In the above configuration, the laser from the laser light source 81 for optical tweezers is divided into two by the polarizing beam splitter-82a. One of the lasers is reflected by the galvanomirror unit 83a, and the other laser is reflected by the galvanomirror unit 83b. The laser beams reflected by the galvanomirror units 83a '83b are superimposed again by the polarizing beam splitter 82b. Thereafter, the superimposed laser beam passes through the lens 84 and the lens 68 and enters the sample cell 19. The fine particles 20 can be held by the laser beam thus incident.

 [0133] Also, since the lasers reflected by the galvanometer mirror units 83a and 83b are condensed at independent locations, the fine particles 20 held by each laser must move independently in the xy plane. Can be.

 Further, the scattered light returning from each fine particle 20 returns to the double beam lower optical tweezer unit 80 as in the configuration of FIG. Of the scattered light that has returned in this way, the light reflected by the beam splitter 88a is condensed by a lens 89a and detected by a quadrant photodetector 90a.

 [0135] On the other hand, of the scattered light returning to the double beam lower optical tweezer unit 80, the light reflected by the beam splitter 88b is condensed by the lens 89b, and then detected by the quadrant photodetector 9 Ob. Is done.

 [0136] Further, the use of the polarization beam splitters 82a and 82b can control the intensity ratio of the beams passing through the two paths at the rotation angle of the half-wave plate 87, and can also control the particle power by the polarization of the reflected light. This is because the reflected light can be separated.

[0137] The half-wave plates 86a and 86b are used to rotate the polarization plane, and the optical power reflected by the polarization beam splitter 82a is transmitted through the polarization beam splitter 82b. Is to adjust.

 [0138] According to the Mariuse's theorem, the following relationship holds between the rotation angle の of the polarization plane and the beam intensity 通過 after passing through the polarization beam splitter 82b.

 [0139] I '= I (cos2 θ + D / 2

 ο

 [0140] Here, I is the light intensity before the polarizing beam splitter 82b. Also, the rotation angle Θ o

 Is a value obtained by setting the angle at which the transmittance becomes maximum to 0 °.

 [0141] Further, when light is incident on particles in a linearly polarized state, most of the reflected light of the particle force retains the original polarization state. By utilizing this property, it is possible to selectively detect scattered light according to the polarization direction.

 Further, the distance between the lens 84 and the lens 64 can be changed in synchronization with the movement of the lens 31 in the same manner as the distance between the lens 64 and the lens 68 described above. The positional relationship between the object 35 and the fine particles can be kept constant.

 According to the double beam lower optical tweezer unit 80 having the above configuration, as shown in FIG. 11, the fine particles 20a and the fine particles 20b attached to both ends of the DNA 91 are separated by the two lasers from the optical tweezer unit. Can be trapped separately. As a result, it is possible to extend the DNA 91 and attach the protein (measurement target) 92-93-94 to the DNA 91 to measure the change in the DNA 91. Note that, when the DNA 91 is expanded, a force acts on each of the fine particles 20a and the fine particles 20b in the directions indicated by arrows A and B in FIG.

 [0144] Protein 92 is a molecule that functions to bend DNA91, and the length of DNA91 changes when such a molecule is attached. The change in the length can be measured by detecting the position of the scattered light from each fine particle 20a'2 Ob.

[0145] The protein 93 is a molecule that is attached and fixed in the vicinity of the protein 92, and the protein 94 is a molecule that binds to and slides on the DNA91. The positions of such proteins 92, 93, and 94 can be measured by irradiating a laser from a confocal microscope and detecting the fluorescence emitted by the fluorescent probe attached to each protein. This makes it possible to observe the relationship between the binding state of the protein 92 and the positional change between the microparticles 20a and 20b, and to observe the slide state of the protein 94 in time series. It will work. In this way, according to the microscope system of FIG. 10, the extension of DNA and the interaction with DNA protein can be measured.

 As described above, the microscope system 1 of the present embodiment includes the optical tweezer unit 2 for holding the fine particles 20, the lens 10 and the actuator 16 for moving the fine particles 20 toward the measurement object, Equipped with a four-segment photodetector 15 that measures the force acting on the object 35 from 20, a confocal unit 3 that allows the object 35 to be magnified and a microscope unit 4! / What is it.

 According to the above configuration, the fine particles 20 held by the optical tweezer unit 2 can be moved by the lens 10 and the actuator 16 and pressed against the measurement target 35. If a chemical substance is immobilized on the fine particles 20, a stimulus by the chemical substance can be given to the measurement object 35. Therefore, the fine particles 20 are held at a position where the fine particles 20 are to be brought into contact with the measurement target 35 by the optical tweezer unit 2, and the fine particles 20 are moved to the measurement target 35 at a predetermined timing using the lens 10 and the actuator 116. By doing so, it is possible to apply a stimulus at a desired timing limited to only a part of the measurement object 35.

 [0148] Further, the force generated on the measurement object 35 by the fine particles 20 being pressed against the measurement object 35 by the lens 10 and the actuator 16 can be measured by using the four-divided photodetector 15. it can. Thereby, the magnitude of the force applied to the measurement target 35 can be evaluated.

 [0149] Further, a change in the shape of the measurement object 35, a change in the internal structure of the measurement object 35, or a change in the molecules constituting the measurement object 35, which is generated when the fine particles 20 are pressed against the measurement object 35, is generated. The spatial distribution can be grasped by enlarging and viewing the object 35 to be measured using the confocal unit 3 and the microscope unit 4. Therefore, it is possible to evaluate whether an appropriate stimulus has been given to the measurement target 35 from the fine particles 20.

Further, by holding the microparticles 20 by the optical tweezer unit 2, the microparticles 20 can be held without bringing the microparticles 20 into contact with the optical tweezers unit 2. Therefore, when moving the fine particles using the lens 10 and the actuator 16, the physical external force is prevented from being applied to the fine particles 20, and the physical characteristics of the fine particles 20 such that the shape of the fine particles 20 is deformed are prevented. Change can be prevented. [0151] Further, as an optical tweezer unit, a double beam lower optical tweezer unit 80 holding a plurality of fine particles by a plurality of laser beams may be used. According to this configuration, a plurality of fine particles can be held by the double beam lower optical tweezer unit 80, so that a plurality of fine particles can be pressed against the measurement target.

 [0152] For example, when proteins 92, 93, and 94 bound to DNA are used as an object to be measured, the microparticles 20a 'and 20b are fixed to both ends of the DNA, and the microparticles 20a' and 20b are fixed by optical tweezers. If immobilized, the relationship between that protein 92, 93, 94 and DNA can be observed. Therefore, the microscope system 1 can be adapted to various measurement methods.

 [0153] Further, the optical tweezer unit 2 uses the lens 10 that is used for the confocal unit 3 and the microscope unit 4 to enlarge the measurement object 35, and the laser beam is incident on the microparticles 20. To retain the fine particles.

 According to the above configuration, since the optical system of the optical tweezer unit 2 and the optical systems of the confocal unit 3 and the microscope unit 4 are configured independently, the holding of the fine particles 20 by the optical tweezer unit 2, The enlargement of the measurement object 35 by the confocal unit 3 and the microscope unit 4 can be adjusted independently. Thereby, the convenience of the microscope system 1 can be improved.

 Further, the four-split photodetector 15 calculates the position displacement amount of the fine particles 20 based on the intensity of the light reflected by the fine particles 20 from the laser light emitted from the optical tweezer unit 2, and calculates the position displacement amount The force acting on the measurement target 35 from the fine particles 20 is measured based on the panel constant indicated by the holding force on the fine particles 20 by the optical tweezer unit 2.

 That is, when the fine particles 20 are held by the optical tweezers unit 2, the holding force on the fine particles 20 is proportional to the amount of displacement of the fine particles 20. Therefore, if the proportional coefficient is calculated in advance as a panel coefficient, the above-mentioned holding force can be calculated from the positional displacement amount of the fine particles 20 and the panel coefficient.

Therefore, in the above configuration, the position displacement amount of the fine particles 20 is obtained using the laser light of the optical tweezer unit 2. That is, since the position displacement amount of the fine particles 20 can be obtained by using the optical system of the optical tweezer unit 2, the configuration for calculating the position displacement amount is simplified. Can. Thus, the configuration of the entire microscope system 1 can be simplified.

Further, by observing the measurement object 35 using a confocal microscope, a tomographic image obtained by optically cutting the measurement object 35 can be obtained. Therefore, it is possible to observe in more detail the change that occurs in the measurement target 35 due to the pressing of the fine particles 20 based on the tomographic image of the measurement target 35 obtained using the confocal microscope.

[0159] Further, a confocal microscope has a pinhole disk 72 having a plurality of pinholes formed on the surface thereof, and a plurality of lenses each of which emits light. A multi-pinhole having a lens disc 71, a pinhole disc 72 and a motor 73 for rotating the microlens disc 71, and an adjusting means capable of adjusting the rotation speed of the two discs 71 and 72 by the motor 73. It is preferable to use the focus unit 70!

 According to the above configuration, by rotating the pinhole disk 72 and the microlens disk 71 using the motor 73, the object 35 to be measured can be scanned using the laser light passing through the lens and the pinhole. Becomes possible.

 If the rotational speed of the disk by the motor 73 is set to a high speed by using the adjusting means, the object 35 to be measured can be scanned at a high speed. Thus, a tomographic image of the measurement target 35 can be obtained at short time intervals. Therefore, it is possible to observe changes occurring in the measurement object 35 due to the pressing of the fine particles 20 in more detail.

 [0162] Furthermore, it is preferable that the confocal microscope be one that observes changes in the measurement object 35 in time series, for example, one that uses a CCD camera 75!

 According to the above configuration, when the measurement object 35 changes over time due to the pressing of the fine particles 20, the change of the measurement object 35 can be viewed in a time series by the CCD camera 75. it can. For example, when the measurement object 35 is a cell and the microparticle 20 is a fixed ligand that stimulates the cell, the response of the cell to the ligand can be viewed in chronological order. As described above, according to the above configuration, more detailed observation of the measurement target 35 caused by the pressing of the fine particles 20 becomes possible.

[0164] Further, the lower optical tweezer unit 60, the quadrant photodetector 15, the lens 64, and the actuator 65 are arranged on the same side of the sample cell 19 on which the measurement object 35 is placed. It may be.

 According to the above-described configuration, the sample cell 19 has a lower optical tweezer unit 60, a quadrant photodetector 15, and a space opposite to the side on which the lens 64 and the actuator 65 are arranged. Resources can be used effectively.

 Further, a lens that changes the spot position of the laser beam of the lower optical tweezers unit 60 based on the amount of movement of the lens 31 used by the confocal unit 3 and the microscope unit 4 to enlarge the object 35 to be measured. With 64 and Actuator 65! /, Preferably! / ヽ

That is, when the lower optical tweezer unit 60 and the confocal unit 3 and the microscope unit 4 are arranged on the same side with respect to the sample cell 19, the laser light of the lower optical tweezer unit 60 Enters the fine particles via the lens 31.

 [0168] Therefore, when the lens 31 moves, the spot position of the laser beam of the lower optical tweezer unit 60 is changed, and the fine particles 20 held and held at the spot position also move. When the fine particles 20 move in this manner, the fine particles 20 cannot be appropriately pressed against the measurement target 35, which may be undesirable in measuring the response of the measurement target 35.

 Therefore, the above configuration includes the lens 64 and the actuator 65 for changing the laser light spot position of the lower optical tweezers unit 60 based on the amount of movement of the lens 31. That is, the movement amount of the lens 31 and the spot position indicate a predetermined relationship. If the spot position is changed by using the lens 64 and the actuator 65 based on this relationship, the fine particles 20 are held at a fixed position, and the positional relationship between the measurement target 35 and the fine particles 20 is kept constant. be able to. Thereby, the fine particles 20 can be stably pressed against the measurement target 35, and the response of the measurement target 35 can be appropriately measured.

[0170] Further, the microscope system 1 preferably includes a cell surgery unit 50 capable of cutting the measurement object 35.

According to the above configuration, by cutting the measurement target 35 using the cell surgery unit 50, the internal structure of the measurement target 35 can be observed in more detail with a confocal microscope. Therefore, the change generated inside the measurement object 35 due to the pressing of the fine particles 20 Can be observed more closely.

 [0172] As described above, according to the microscope system 1, the positional displacement of the fine particles in the X, y, and z directions, the time-series change of the stimulus to the measurement target, and the force acting on the measurement target include: It can measure 5-dimensional physical quantities. From this point, the microscope system 1 of the present embodiment can be called a “five-dimensional microscope”.

 [0173] The present invention is not limited to the above-described embodiment, and various changes can be made within the scope shown in the claims. For example, as the holding means, a micromanipulator may be used other than the optical tweezers, or a device for fixing the fine particles using a magnetic field may be used. As observation means, other than a confocal microscope, a device that uses stereoscopic microscopy, a CT microscope, or a device that removes blurred images using a deconvolution method and performs three-dimensional observation (Non-patent Document 4; Biological spectroscopy, seeing invisible objects beyond spectroscopy ”, spectroscopy Society of Japan, Measurement Methods Series 21”, published by the Society of Research Publishing, June 30, 1991).

According to the measurement system of the present invention, the fine particles held by the holding means can be moved by the moving means and pressed against the measurement object. In addition, if a chemical substance is immobilized on the fine particles, stimulation by the chemical substance can be applied to the object to be measured. Therefore, by holding the fine particles at a position where the fine particles are to be brought into contact with the measurement object by the holding means, and further moving the fine particles to the measurement object at a predetermined timing using the moving means, the measurement object can be obtained. Thus, it is possible to provide a stimulus at a desired timing by limiting the stimulus to only a part of the stimulus.

 [0175] Further, the amount of stimulus generated in the measurement target object when the fine particles are pressed against the measurement target object by the moving means can be measured by using the measurement means. Accordingly, an effect is obtained that the magnitude of the force applied to the measurement target can be evaluated.

[0176] Further, the shape change of the measurement object, the change in the internal structure of the measurement object, or the spatial distribution of molecules constituting the measurement object caused by the pressing of the fine particles against the measurement object are as follows. It can be grasped by enlarging and observing the measurement object using observation means. Therefore, it is possible to evaluate whether an appropriate stimulus has been given to the object to be measured by the fine particle force! [0177] It should be noted that the specific embodiments or examples made in the section of the best mode for carrying out the invention merely clarify the technical contents of the present invention, and such specific Various modifications can be made within the spirit of the present invention, which should not be construed in a narrow sense by limiting only to the examples, and the claims described below. Industrial potential

An example of a field of use of the microscope system having the above configuration will be described below. First, the state of a disease such as diabetes can be evaluated by measuring the hardness of red blood cells using the microscope system configured as described above.

[0179] To measure the hardness of red blood cells, the red blood cells are fixed on a glass substrate, and fine particles for applying a force to the red blood cells are dispersed in a buffer solution. Then, the microparticles are captured using the optical tweezers of the microscope system 1 and pressed against the red blood cell surface. At this time, the deformation of red blood cells is measured three-dimensionally with a confocal microscope while measuring the amount of displacement of the fine particles.

 [0180] As a result, the force applied to the surface of the red blood cell can be obtained as the displacement amount force of the fine particles, and the local stress distribution can be estimated from the deformation of the red blood cell at that time. From this estimation result, the mechanical properties of red blood cells can be quantitatively evaluated.

 [0181] Similarly, by examining the response of cells to a stimulus using a microscope system, one can also examine changes in cells due to lesions. Examples of the stimulus given to the cell include a stimulus by a chemical substance, a physical stimulus such as pressing a cell, and a stimulus by a local temperature change.

 [0182] When stimulating a cell with a chemical substance, the chemical substance is immobilized on the surface of the particle. The chemical substance is contained inside the particle, and the particle is caught by optical tweezers and applied to the cell as a sample. The stimulus is given by contact with the skin. The response to the stimulus at this time is observed through a microscope optical system.

[0183] Physical stimulation can be achieved by capturing fine particles for applying a stimulus with optical tweezers, pressing the fine particles against cells, or applying a lateral force. It is. In addition, applying a stimulus due to a local temperature change can be achieved by irradiating light of a specific wavelength with fine particles containing a substance that causes a temperature rise. This can be achieved by contacting the microparticles with cells and irradiating the microparticles with light of a specific wavelength.

 The results of examining the response of cells to stimuli in this way can be used to examine cellular responses to environmental hormones, to investigate cellular responses to drugs in the field of drug discovery, to screen for pharmacological substances, and to extract for medical diagnostic purposes. Can be applied to the cytology of isolated cells

Claims

The scope of the claims

 [1] holding means for holding fine particles,

 Moving means for moving the fine particles toward the measurement object,

 A measurement system comprising: a measurement unit configured to measure an amount of a stimulus acting on the measurement target from the fine particles; and an observation unit configured to enlarge and view the measurement target.

 [2] The measurement system according to claim 1, wherein the holding means is an optical tweezer.

 [3] The measurement system according to claim 2, wherein the optical tweezers hold a plurality of fine particles by a plurality of laser beams.

[4] The optical tweezers use a second lens different from the first lens used by the observation means to enlarge the object to be measured, and apply laser light to fine particles to hold the fine particles. The measurement system according to claim 2 or 3, wherein:

[5] The measuring means is

 Based on the intensity of the laser light emitted from the optical tweezer unit reflected by the fine particles, the amount of positional displacement of the fine particles is calculated,

 5. The method according to claim 1, wherein the magnitude of the force as the amount of the stimulus is measured based on the positional displacement amount and a panel constant indicated by the holding force of the optical tweezers on the fine particles. The measuring system according to paragraph 1.

[6] The measuring system according to any one of claims 1 to 5, wherein the observation means is a confocal microscope.

[7] The confocal microscope is

 A pinhole disk having a plurality of pinholes formed on its surface,

 A microlens disc having a plurality of third lenses that emit light in each of the plurality of pinholes,

Rotating means for rotating the pinhole disk and the microlens disk; and adjusting means capable of adjusting the rotational speed of the disk by the rotating means. 7. The measuring system according to claim 6, wherein:

[8] The measurement system according to any one of claims 1 to 7, wherein the observation means is for observing a change of the measurement object in a time series.

[9] The apparatus according to claim 1, wherein the holding means, the measuring means, and the observation means are arranged on the same side with respect to a mounting table on which the object to be measured is mounted. Measurement system.

[10] The holding means is an optical tweezer,

 The observing means includes a spot position changing means for changing a spot position of the laser beam of the optical tweezers based on a movement amount of a first lens used for enlarging the measurement object. Measurement system according to range 9.

[11] The measurement system according to claim 1, further comprising a cutting means capable of cutting the object to be measured.

12. The measurement system according to claim 11, wherein the cutting means cuts the measurement object by irradiating the measurement object in a state where the pulse laser light is focused.