JP3766870B2 - Micro force measuring device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a micro force measuring apparatus that measures, over time, a mechanical force acting between micro particles such as protein molecules and a predetermined base substance.
[0002]
[Prior art]
Conventionally, for example, a minute glass probe for measuring the magnitude of a minute force generated when a motor protein molecule as a minute particle interacts with a protein filament as a base substance with an accuracy of pN (piconeuton) A method using optical tweezers has been known. These use a small glass probe tip or optical tweezers as a small spring balance to fix a single motor protein molecule on the particles captured by the micro glass probe tip or optical tweezers. When the protein filament is fixed on the surface and the two are brought close to each other, when the interaction works between them, the displacement of the particles captured by the tip of a small glass probe or optical tweezers based on the movement of the motor protein is observed, By measuring this displacement, it is possible to measure the change over time in the strength of the force acting between the motor protein molecule and the protein filament (see, for example, Patent Document 1 and Non-Patent Document 1). ).
[0003]
Also, by using the laser radiation pressure, feedback control of the position of the probe in the direction perpendicular to the surface of the object to be observed allows the direction perpendicular to the surface of the object to be observed while maintaining a constant distance from the surface of the object to be observed. An intermolecular force microscope that performs non-contact measurement of the attractive force and repulsive force is known (see, for example, Patent Document 2).
[0004]
However, these methods do not consider means for measuring the force in the horizontal direction by eliminating the displacement caused by thermal fluctuations and external forces of the probe and particles, and therefore, the relative positions between the minute particles are not counted. The problem remains that the horizontal force cannot be detected while controlling in nanometer order. Therefore, in order to solve the problem, the applicant of the present invention is an apparatus capable of controlling the relative position in the horizontal direction between minute particles such as motor proteins and protein filaments and the base material in the order of several nanometers. A probe position for performing feedback control so as to statically fix the probe that fixes a minute particle and the probe whose position changes as the minute particle moves to a predetermined target position using the radiation pressure of the laser for light pressure A control mechanism and a measuring means for measuring and recording the laser radiation output of the light pressure laser over time have been developed. (For example, see Patent Document 3)
[0005]
[Patent Document 1]
JP 9-43434 A
[Patent Document 2]
Japanese Patent Laid-Open No. 7-12825
[Patent Document 3]
Japanese Patent Application No. 2001-308744
[Non-Patent Document 1]
TREND in Biotechnology vol.19 no.6 June 2001 P211-P216
[0006]
[Problems to be solved by the invention]
[0007]
By the way, when measuring a minute force with the above-mentioned apparatus, it is necessary to fix the minute particles to the probe tip in the previous stage of the operation related to the measurement. In this case, the probe is attached to a plurality of minute particles. And then fixed by a predetermined chemical method. As a result, as shown in FIG. 15, the base material (in 1030) remains in a state where a plurality of minute particles (indicated by 1029) are attached to the probe (indicated by 1000a) of the probe (indicated by 1000). In the conventional apparatus, the number of fixed fine particles cannot be confirmed, so the measured value per minute particle is It is difficult to get instantly.
[0008]
Therefore, in order to solve such disadvantages, it was possible to confirm the number of fine particles fixed to the probe, and it was possible to obtain a measurement value of force per minute particle A micro force measuring device is provided.
[0009]
[Means for Solving the Problems]
That is, the present invention is for measuring a mechanical force acting between a minute particle and a predetermined base substance, and includes a probe that fixes the minute particle, and the movement of the minute particle. A probe position control mechanism that feedback-controls the probe whose position changes with the stationary pressure to a predetermined target position using the radiation pressure of the light pressure laser, and measures and records the radiation output of the light pressure laser over time The micro force measuring device comprises a particle number measuring mechanism for measuring the number of the micro particles fixed to the probe.
[0010]
The “measuring the number immobilized on the probe” is not limited as long as it can directly or indirectly measure the number of fine particles immobilized on the probe. For example, the particle is visually observed. And those that are judged by observing or measuring a predetermined element having a correlation with the number of particles.
[0011]
In such a case, since the number of fine particles fixed to the probe can be confirmed, only one fine particle can be fixed to the probe. In other words, the measured value of the force acting between one minute particle and the base material can be obtained immediately without analyzing or confirming whether the obtained value is really the force of one minute particle. It becomes like this.
[0012]
As the particle number measurement mechanism, a mechanism having a total reflection fluorescence observation mechanism is suitable. This is because, according to the total reflection fluorescence observation mechanism, the process of fixing a minute substance to the probe can be directly observed in real time in a visual manner. In addition, it is possible to easily configure other substances such as a base substance to be observed, and in this way, the positional relationship between the minute substance and the base substance can be adjusted. These interactions can be measured in an appropriate state.
[0013]
Moreover, as a preferable specific aspect of the total reflection fluorescence observation mechanism, there is one in which an excitation light laser is irradiated from the objective lens to the observation object surface and totally reflected. If it is such, it will become easy to ensure the installation space of a probe above an observation object surface.
[0014]
It is preferable that at least a region of the probe irradiated with the light pressure laser is formed of a non-metallic dielectric. Even when the irradiated area is irradiated with light from a laser for light pressure, heat generation due to free electron vibration does not occur, so that accurate measurement values can be obtained with high accuracy without deformation or deterioration of the probe. It is. Thus, if the measurement value as accurate as possible can be obtained, the usefulness of the apparatus of the present invention that enables measurement of the force of one minute particle can be greatly improved. In addition, it is possible to increase the number of times the probe can be used and the time to prevent an increase in cost due to replacement of parts. Note that examples of the nonmetallic dielectric include glass, quartz glass, quartz, silicon, and diamond.
[0015]
As a preferred specific embodiment of such a probe, a probe for fixing minute particles, a light irradiation unit connected to the probe and irradiated with the laser for light pressure, and a support unit for supporting the light irradiation unit The thing which comprises and is mentioned.
[0016]
As a light irradiation part, what was comprised with plate glass and what was comprised with the prism are mentioned.
[0017]
As a specific aspect of the support portion, a base material may be fixed to the observation object surface and configured to be movable in the horizontal direction with respect to the observation object surface. In such a case, the light irradiation part and the support part are formed so as to be an integrated plate-like body, and the integrated light irradiation part and the support part are installed in a direction perpendicular to the observation target surface. In this way, the manufacturing can be simplified. Further, in order to correspond to the movement of the minute particles fixed to the probe in a direction perpendicular to the observation target surface, the support portion is configured to be movable in the vertical direction with respect to the observation target surface. What should I do?
[0018]
In order to simplify and simplify the apparatus, a laser radiation pressure may be applied by irradiating a laser for light pressure from an objective lens.
[0019]
As a specific configuration of the probe position control mechanism, a position detection light irradiating means for irradiating light to the probe, a projecting means for projecting an interference image by the light irradiation on a split photodiode, and a position of the probe by the interference image And a detecting means for detecting and measuring the position of the probe as a probe position detecting means, and further comprising an adjusting means for adjusting the intensity of the laser for light pressure applied to the probe from the detection result of the detecting means.
[0020]
Regarding the suppression of probe fluctuation due to heat, in the probe position control mechanism, the fluctuation of the probe is converted to a mean square displacement and is monitored and controlled to be held at 0.6 nanometers or less. It is possible to prevent the probe from fluctuating.
[0021]
In addition, a probe with a constant spring constant is connected to the fixture to enable measurement regardless of whether fine particles move in the forward or reverse direction relative to the irradiation direction of the laser for light pressure. It is desirable to load the probe with a radiation pressure of an arbitrary intensity and add an offset function so that dynamic equilibrium can be maintained.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
[0023]
This minute force measuring device fixes minute particles such as proteins to the probe 1 and measures the mechanical force acting between the minute particles and the base substance fixed to the observation object surface 2a. As shown in FIG. 1 and FIG. 2, the probe 1 and the probe 1 whose position changes with the movement of the minute particles are arranged as shown in FIG. It has a function as a probe position control mechanism A that performs feedback control so as to be stationary and fixed at a predetermined target position using the radiation pressure, and a measuring means B that measures and records the radiation output of the optical pressure laser over time.
[0024]
More specifically, as shown in FIGS. 3 to 6, the probe 1 first irradiates light that is a probe 1 a that fixes minute particles, and a region that is connected to the probe and is irradiated with the light pressure laser. And a thin plate-shaped cantilever 1d that functions as a support portion 1c that supports the light irradiation portion 1b.
[0025]
In this embodiment, the probe 1a employs a ZnO whisker, and fine particles such as protein are fixed to the tip of the probe 1a. As a fixing method, gold is attached to the probe 1a and a part of a protein, which is a fine particle, is modified with an SH group, and nickel is attached to the probe 1a and a His tag is fused to the protein. And a method of binding by the interaction of avidin and biotin.
[0026]
Further, the cantilever 1d is formed to have a length of 200 μm, a width of 20 μm, and a thickness of 100 nm, for example. The free end side to which the probe 1a is attached is used as the light irradiation unit 1b, and the base end is attached to an arbitrary fixing device (not shown). The side also serves as the support portion 1c. The spring constant is set to be about 0.1 pN / nm so that the spring constant can be moved in the horizontal direction with respect to the observation target surface 2a, and as shown in FIGS. 5 and 6, the observation target surface 2a. Is attached to the fixing device in a cantilevered manner. Note that the observation target surface 2a is a place where minute particles fixed to the probe 1a move, and a slide glass 2 is usually used. And in this embodiment, this cantilever 1d which functions as the light irradiation part 1b and the support part 1c is comprised with the plate-shaped glass which is a nonmetallic dielectric material.
[0027]
The probe position control mechanism A includes an irradiation unit A1 for irradiating the probe 1 with a laser for light pressure, a position detection irradiation unit A2 for irradiating the probe 1 with light for position detection, and the position detection for the split photodiode. A projecting means A3 for projecting an interference image by light irradiation of the irradiating means A2, a detecting means A4 for detecting and measuring the position of the probe 1 from the interference image, and a light pressure laser for irradiating the probe 1 from the detection result by the detecting means A4. And adjusting means A5 for adjusting the strength of the lens.
[0028]
The irradiation means A1 adjusts the irradiation intensity of the light beam of the first laser light source 3 that irradiates the laser for light pressure by an EOM (electro-optic modulator) 4, passes through the laser lens for light pressure 5, and passes through the first light source. The light is reflected by one reflecting mirror 6, is incident on a first objective lens 7 corresponding to the objective lens according to claim 11, is condensed, and is irradiated onto the probe 1. Although not shown in the illustrated example, the laser beam output from the EOM 4 may be reflected by a plurality of condensing lenses or reflecting mirrors and incident on the first objective lens 7. In this embodiment, a YAG (Yttrium Aluminum Garnet) laser (1064 nm, 500 mW) having a wavelength in the infrared region in which almost no absorption is observed in the glass is employed as the light pressure laser. Moreover, it sets so that it may irradiate to the diagonal direction with respect to the surface of the light irradiation part 1b which makes | forms plate shape, and the incident angle is about 80 degrees in this embodiment. As shown in FIG. 7, in P-polarized light and S-polarized light, the angle at which the greatest force can be obtained is selected from the relationship between the incident angle and the force applied to the glass cantilever 1d. In addition, as shown in FIG. 8, it has confirmed that the position of the probe 1 changes reliably and shows a reproducible result by the presence or absence of this optical pressure laser. Furthermore, as shown in FIG. 9, it has also been confirmed that there is a proportional relationship between the intensity of the laser for light pressure and the force applied to the probe 1. In addition, as shown in FIG. 10, the micro force measuring device of the present embodiment provides an offset function in which a light pressure of an arbitrary intensity is loaded on the probe 1 to bring the probe into a predetermined target position by dynamic equilibrium. As a force required for the offset, the radiation pressure of the laser for optical pressure is set to be sufficiently larger than the force of the fine particles fixed to the probe 1. For example, when measuring the movement of a protein as fine particles, a force larger than the force of this movement may be used, and therefore, it should be set in the range of more than 8 pN, preferably 25 pN.
[0029]
As shown in FIG. 6, the position detection light irradiation means A2 spreads the light beam of the second laser light source 8 for position detection by the beam expander 9 and then reflects it by the second reflecting mirror 10. The light is incident on the first objective lens 7, condensed, focused (crossed) on the probe 1 and irradiated. As shown in FIG. 1, the light beam from the second laser light source 8 passes through beam splitters 20 and 23 (described later), and the transmitted light is used. Further, the position detection laser needs to be configured so as not to have a special effect on the probe 1. In this embodiment, a YAG laser (532 nm, 100 μW) is employed.
[0030]
The projection means A3 causes the position detection laser beam that has passed through the light irradiation unit 1b to be incident on the second objective lens 12, reflected by the third reflecting mirror 11, and probe interference within the two-divided photodiode 13 that is a divided photodiode. It is configured to obtain an image.
[0031]
The detection means A4 converts the interference image into an electric signal as a position signal by a position detection amplifier 16 having an IV converter 14 and a differential amplifier 15 connected to the two-divided photodiode 13, and further converts A- The digital signal is converted by the D converter 17 and the output signal is integrated and recorded in the data recording computer 18 for monitoring.
[0032]
The adjustment means A5 is configured to convert the position signal into a feedback signal by the feedback circuit 19 and transmit the signal to the EOM 4 to adjust the intensity of the optical pressure laser, thereby fixing the probe 1 stationary. At this time, as shown in FIG. 3, the radiation pressure of the laser for optical pressure is applied in the opposite direction to the force applied by the fine particles by feedback control, and the probe 1 is made to stand still on the appearance. Here, the movement of minute particles fixed to the probe 1 and the fluctuation due to heat as the background are generated in the probe 1. By inputting the position signal to the feedback circuit 19 and following the target value signal, the fluctuation of the probe 1 is reduced. It is set so that it is suppressed to 0.6 nanometer or less in terms of root mean square displacement. The interference fringes change when the position of the probe 1 is displaced, but it is known that the amount of change changes with a certain correlation with the position change of the probe 1. Position measurement is possible.
[0033]
The measuring means B measures the radiation output over time by the computer 18 connected to the first laser light source 3 or the EOM 4 or a dedicated control device (not shown), etc., and mechanically loads the probe 1 indirectly. It can be measured.
[0034]
Therefore, the micro force measuring apparatus of the present embodiment further includes a total reflection fluorescence observation mechanism C which is a particle number measuring mechanism for measuring the number of the micro particles fixed to the probe 1.
[0035]
The total reflection fluorescence observation mechanism C is based on the principle of a so-called total reflection fluorescence observation microscope that observes a fluorescent substance excited in an evanescent field generated by totally reflecting excitation light with respect to a predetermined surface such as an observation target surface. Is based. In the present embodiment, the number of minute particles fixed to the probe 1a can be observed in a recognizable manner, and the base material can be observed. The predetermined fluorescent material labeled on the minute particles and the base A laser beam having a wavelength for exciting a fluorescent substance different from the fluorescent substance labeled on the substance is irradiated at an incident angle set so as to totally reflect the observation target surface 2a, and then from the observation target surface 2a. An evanescent field is formed in the range of about 100 nm.
[0036]
More specifically, first, the excitation light for observing fine particles uses the second laser light source 8 that constitutes the position detecting irradiation means A2, that is, a YAG laser (532 nm, 500 μW) is used. ing. Then, the light beam from the second laser light source 8 is branched by the beam splitter 20, the reflected light is spread by the beam expander 21, passed through the condenser lens 22, reflected by the mirror MR, and again by the beam splitter 23. The light is reflected, returned to the light of the total reflection optical system, then reflected by the second reflecting mirror 10, passed through the first objective lens 7, and irradiated on the observation object surface 2 a from below. Here, by adjusting the condensing lens 22 so as to condense on the rear focal plane of the first objective lens 7, parallel light is obtained on the observation object surface 2a, and the position of the mirror MR is adjusted, An incident angle for total reflection with respect to the observation object surface 2a is obtained. In order to selectively use the second laser light source 8 as a position detection light source or as an excitation light source, for example, a shutter (not shown) can be installed to adjust irradiation on / off. I have to.
[0037]
On the other hand, a blue laser (473 nm, 500 μW) is used as the excitation light for observing the base material, and the light beam of the third laser light source 24 for excitation is passed through the beam expander 25 and the condenser lens 26. The object surface 2 a is reflected from the lower side through the first objective lens 7 after being reflected by the third reflecting mirror 27.
[0038]
Then, the irradiation areas of these respective excitation lights are imaged by, for example, the CCD 28, and the state of the fluorescence is displayed as an image on a monitor (not shown).
[0039]
As shown in FIG. 11, the force acting between the motor protein (kinesin) 29, which is a fine particle, and the filament protein (microtubule) 30, which is a base substance, is measured by the micro force measuring apparatus configured as described above. The operation of the case will be described.
[0040]
First, a process from fixing the motor protein 29 to the probe 1a of the probe 1 and measuring the micro force will be described. The motor protein 29 is previously labeled with a predetermined fluorescent material such as Cy3, and the filament protein 30 is previously labeled with a predetermined fluorescent material such as Alexa488. When excitation light from the third laser light source 24 is irradiated onto the observation object surface 2a so that the position of the fluorescently labeled filament protein 30 fixed to the observation object surface 2a can be confirmed, the CCD camera 28 is excited. The state of fluorescence by light is imaged and the image is displayed on the monitor. Next, when the second laser light source 8 is irradiated as a light source for excitation light so as to totally reflect the observation target surface 2a, the CCD camera 28 similarly captures the fluorescence state of the excitation light. The image is displayed on the monitor. Thereby, the position of the motor protein 29 can be confirmed. The probe 1 scans according to a predetermined program and picks up the motor protein 29 with the probe 1a. At this time, if the excitation light from the second laser light source 8 is continuously irradiated and it is observed that the fluorescent spot on the probe 1a disappears in one step, it can be confirmed that one molecule is fixed. Become. Thereafter, a light beam is emitted from the third laser light source 24 and the fluorescence state based on the excitation light is displayed on the monitor, so that the probe 1a is moved to the filament protein 30 while confirming the position of the filament protein 30 on the screen. 30 can be deposited.
[0041]
After the measurement preparation is completed in this way, the second laser light source 8 is switched to function as a position detection for the probe 1 by the input of the measurement start signal, and the first laser light source 3 for light pressure is used. Irradiation starts. Specifically, the laser beam from the first laser light source 3 is applied to the surface of the light irradiation part 1b of the cantilever 1d through the first objective lens 7 in an incident angle of 80 ° and a diameter of about 20 μm. Here, when the light pressure laser is irradiated to the light irradiation portion 1b made of glass (indicated by F1 in the figure), as shown in FIGS. 3 and 4, a part of the glass is irradiated at a rate depending on the incident angle. Are reflected (indicated by F2 in the figure), partially refracted (indicated by F3 in the figure), and transmitted through (indicated by F4 in the figure).
[0042]
On the other hand, the laser beam output from the second laser light source 8 is spread by the beam expander 9, reflected by the second reflecting mirror 10, incident on the first objective lens 7 and condensed, and the position of the probe 1. Is detected. The position detection laser that has passed through the probe 1 enters the second objective lens 12 and is reflected by the third reflecting mirror 11. Then, a probe interference image is obtained in the two-divided photodiode 13 reflecting the position of the probe 1. Here, the probe 1 is randomly displaced due to thermal fluctuation, but this is detected as the movement of the interference image (FIGS. 12 and 13). The interference image is converted into an electric signal as a position signal by the position detection amplifier 16, digitized by the A / D converter 17, and input to the computer 18. The computer 18 converts the background due to thermal fluctuation or the like into a mean square displacement and calculates. The position signal enters the feedback circuit 19 and is converted into a feedback signal. This feedback signal shifts to the EOM 4 and the irradiation intensity of the first laser light source 3 is adjusted according to the feedback signal.
[0043]
Then, regardless of the generation of force by the motor protein 29 fixed to the probe 1a by the radiation pressure of the light pressure laser adjusted by the feedback signal, the probe 1 is apparently fixed stationary. At this time, the displacement of the probe due to the thermal fluctuation when the feedback shown in FIG. 12 is not applied can be controlled and fixed with an accuracy of about 0.6 nanometer by feedback control as shown in FIG. Can be controlled in the same way as In this embodiment, since the offset function is added, as shown in FIG. 10, when the motor protein 29 fixed to the probe 1 moves in the direction opposite to the light pressure laser irradiation direction, radiation is performed. The strength applied to the pressure corresponds to the force obtained by adding the force applied to the offset and the force accompanying the movement of the motor protein 29. On the other hand, when the motor protein 29 moves in the same direction as the light pressure laser irradiation direction, the radiation pressure of the light pressure laser corresponds to a force obtained by subtracting the force accompanying the movement of the motor protein 29 from the force acting on the offset. It will be.
[0044]
Then, the force acting between the motor protein 29 and the filament protein 30 is obtained by using the spring constant of the cantilever 1d to determine the relationship between the feedback signal and the radiation pressure of the optical pressure laser with respect to the probe 1 and analyzing the feedback signal. Can be obtained.
[0045]
Since the micro force measurement apparatus of the present embodiment described above incorporates the total reflection fluorescence observation mechanism C, which is a particle number measurement mechanism, the process of fixing the motor protein (kinesin) 29, which is a minute substance, to the probe 1a. Can be directly observed and confirmed in real time, and only one molecule of the motor protein 29 can be fixed to the probe 1a. As a result, the force (sliding force) per molecule of the motor protein 29 can be measured appropriately.
[0046]
Further, since the total reflection fluorescence observation mechanism C of the present embodiment can also observe the filament protein 30 as a base material, the probe in a state where the motor protein 29 is fixed while checking the position of the filament protein 30. 1a can be deposited at an appropriate position. That is, the force acting between the motor protein 29 and the filament protein 30 can be accurately measured.
[0047]
Further, both the excitation laser for observing the motor protein 29 and the excitation laser for observing the filament protein 30 are irradiated from the first objective lens 7 to the observation object surface 2a from below and totally reflected. Since it comprised in this way, the space which arrange | positions a cantilever above the filament protein 30 mounted in the observation object surface 2a can be ensured easily, and it becomes easy to design and manufacture an apparatus.
[0048]
In the present embodiment, since the second laser light source 8 that is a component of the probe position control mechanism A functions also as an excitation laser light source that constitutes the total reflection fluorescence observation mechanism C, the number of parts is reduced. The cost can be reduced and the device can be made compact.
[0049]
In addition, since the cantilever 1d having a function as the light irradiation unit 1b irradiated to the laser for light pressure is made of glass, compared to a cantilever coated with metal, deformation due to generation of heat is prevented, Accurate data can be obtained. In addition, since the apparatus of this embodiment can appropriately measure the force per minute particle as described above, the measurement value obtained by adopting such a cantilever 1d made of glass is used. The improvement in accuracy has a synergistic effect, and can provide highly reliable data that has never existed before. Further, by preventing deformation due to heat, the probe 1 can be used for a long time, and cost reduction can be expected. Furthermore, since glass is employed as a non-metallic dielectric, it is easy to obtain and process.
[0050]
In this embodiment, the light pressure laser is adjusted in intensity by the EOM 4 as described above, and reaches the probe 1 via the light pressure laser lens 5, the first reflecting mirror 6, and the first objective lens 7. I have to. The position detection laser causes the light beam from the position detection laser light source 8 to reach the probe 1 via the beam expander 9, the second reflecting mirror 10, and the first objective lens 7. Furthermore, the mechanism for total reflection observation of minute particles also branches the second laser light source 8 so that the beam expander 21, the condensing lens 22, the second reflecting mirror 10, and the first objective lens 7 are separated. Through the observation object surface 2a. The excitation laser for observing the base material with total reflection also observes the light beam of the third light source 24 through the beam expander 25, the condensing lens 26, the third reflecting mirror 27, and the first objective lens 7. It is made to reach the object surface 2a. That is, since all of these optical systems that can be handled relatively easily and flexibly are employed, a plurality of light sources having different wavelengths can be easily connected. In addition, since all of the laser for pressure, the laser for position adjustment, and the laser for excitation are set so as to pass through the first objective lens 7, the configuration of the apparatus can be simplified and it can be prevented from becoming bulky.
[0051]
In addition, since the offset function is added to the probe 1, measurement can be performed even if minute particles fixed to the probe 1 move in any direction left or right with respect to the observation target surface 2a.
[0052]
Further, in the micro force measuring apparatus according to the present embodiment, the irradiation means A1 for irradiating the probe 1 with the laser for optical pressure is configured to include the 500 mW YAG laser and the EOM 4, so that feedback control is performed using a maximum force of 30 pN. It has also been clarified through experiments that can be performed at 0.6 nm rms. That is, since control can be performed with very high accuracy, the force of minute particles can be measured more accurately.
[0053]
The present invention is not limited to the above embodiment.
For example, the total reflection fluorescence observation mechanism uses the above-mentioned blue laser as excitation light to observe a single molecule of a motor protein, and a YAG laser or other green laser as excitation light to observe the filament protein. A laser having a wavelength different from that of the embodiment may be used.
[0054]
Further, the total reflection fluorescence observation mechanism may further be able to observe ATP (fluorescently labeled) when the motor protein moves. If it is such, the relationship between the mechanical force of a motor protein and the chemical reaction of ATP can be measured.
[0055]
Further, the total reflection fluorescence observation mechanism may be capable of observing only minute particles.
[0056]
The mechanism for measuring the number of particles is to bind the motor protein to a small bead at a certain ratio, and plot the ratio of the bead driven by the motor protein as a function of the bead: motor protein ratio when measuring the microforce. Or the number of particles may be determined. In other words, when the ratio is small enough, that is, when the motor protein is not bound to most of the beads, the moving bead is observed that only one molecule of the motor protein is immobilized. is there.
In addition, a particle number measurement mechanism may be used in which the micro force to be measured is quantized and the minimum unit force is based on a single molecule.
[0057]
The probe may be such that only the irradiated region is made of glass, or the entire probe including the probe may be made of glass. The material constituting the light irradiation part is not limited to glass as long as it is a non-metallic dielectric material, and may be, for example, quartz glass, quartz, silicon, or diamond.
[0058]
Further, the probe may be such that the light irradiation part and the support part are formed separately. In addition, the material constituting the probe is not limited to the non-metallic dielectric as described above, and may be a probe including a light irradiation part and a support part (cantilever) coated with metal on glass, for example.
[0059]
Further, as shown in FIG. 14, a probe 100 including a probe 100a, a light irradiation unit 100b constituted by a prism, and a columnar or prismatic support 100c separate from the light irradiation unit 100b. It may be such that it has. The diameter of the support part 100c is set smaller than that of the light irradiation part 100b, and the support part 100c is movable in the horizontal direction with respect to the observation object surface 2a and is movable in the vertical direction. In addition, the same code | symbol is attached | subjected and shown to the structure similar to the said embodiment. Even with such a probe 100, it is possible to suppress the generation of heat due to irradiation of the laser for light pressure and to give an accurate measurement value. In addition, as shown in the figure, the incident light is indicated by F1 and the reflected light is indicated by F2, and if it is a prism, the light pressure laser can be totally reflected, so that the irradiation intensity of the light pressure laser necessary for measurement can be suppressed. Is possible. In addition, in the case of such a probe 100, when a force in a direction perpendicular to the observation object surface 2a when the fine particles move is generated, the force is absorbed by the movement of the support portion 100c. Therefore, measurement can be performed without crushing minute particles fixed to the probe 1a.
[0060]
The fine particles are not limited to proteins, but may be other fine particles such as DNA, RNA, ATP, cells, and the like.
[0061]
In addition, the specific configuration of each part is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.
[0062]
【The invention's effect】
The present invention is implemented in the form as described above, and has the following effects.
[0063]
That is, according to the micro force measuring apparatus of the present invention, since the particle number measuring mechanism is provided, the number of micro particles fixed to the probe can be confirmed, and only one micro particle is fixed to the probe. Is possible. In other words, it is possible to immediately obtain a measurement value of the force acting between one minute particle and the base material without analyzing or confirming whether the obtained value is really the force of one minute particle. Can do.
[0064]
In particular, when the particle number measurement mechanism is equipped with a total reflection fluorescence observation mechanism, the fixation process of a minute substance to a probe can be directly observed in real time, so only one minute particle can be observed. Can be securely fixed to the probe.
[Brief description of the drawings]
FIG. 1 is a device configuration diagram showing an embodiment of the present invention.
FIG. 2 is a functional configuration diagram showing the embodiment.
FIG. 3 is an explanatory diagram showing a state in which the probe is irradiated with a light pressure laser in the embodiment.
FIG. 4 is an explanatory view showing a state in which the probe for light pressure in the embodiment is irradiated to the probe through the surface of the observation object.
FIG. 5 is a partial perspective view showing a state of a probe and an observation object surface in the same embodiment.
FIG. 6 is an explanatory diagram illustrating a state in which the probe is irradiated with a position detection laser in the embodiment.
FIG. 7 is a graph showing the relationship between the incident angle of the optical pressure laser to the probe and the force acting on the probe in the same embodiment;
FIG. 8 is a graph showing a result of changing the position of the probe due to the radiation of the light pressure laser when the intensity of the light pressure laser is changed in the embodiment.
FIG. 9 is a graph showing the relationship between the light pressure laser intensity and the radiation pressure applied to the probe in the same embodiment;
FIG. 10 is an explanatory diagram showing an offset function in the embodiment.
FIG. 11 is a diagram showing a case where a force acting between a motor protein and a filament protein is measured in the same embodiment.
FIG. 12 is a graph showing a change with time of displacement due to thermal fluctuation of the probe when feedback control is not performed in the embodiment.
FIG. 13 is a graph showing a change with time of displacement due to thermal fluctuation of the probe when feedback control is applied in the embodiment.
FIG. 14 is a perspective view showing the main part of another embodiment of the present invention.
FIG. 15 is a diagram showing a state during measurement by a conventional apparatus.
[Explanation of symbols]
1 ... Probe
1a ... Probe
1b: Light irradiation part
1c ... support part
2a: Object surface to be observed
7 ... Objective lens (first objective lens)
13: Split photodiode (dual photodiode)
29 ... fine particles (motor protein)
30 ... Base material (filament protein)
100: Probe
100a ... probe
100b ... Light irradiation part
100c ... support part
A: Probe position control mechanism
A2 ... Irradiation means for position detection
A3 ... Projection means
A4 ... Detection means
A5 ... Adjustment means
B ... Measurement means
C ... Particle number measurement mechanism (total reflection fluorescence observation mechanism)

Claims (14)

It is for measuring the mechanical force acting between a minute particle and a given base material,
A probe fixing the fine particles;
A probe position control mechanism that performs feedback control so that the probe whose position changes with the movement of the minute particles is stationary and fixed to a predetermined target position using a radiation pressure of a laser for light pressure;
In a micro-measuring device having measuring means for measuring and recording the radiation output of the laser for light pressure over time,
Comprising a particle number measuring mechanism for measuring the number of the fine particles fixed to the probe ;
A region irradiated with at least the light pressure laser of the probe is formed of a non-metallic dielectric,
A part of the laser for light pressure transmits a region irradiated to the laser for light pressure of the probe . 2. The micro force measuring apparatus according to claim 1, wherein the light pressure laser is a YAG laser. The micro force measuring apparatus according to claim 1 or 2 , wherein the particle number measuring mechanism includes a total reflection fluorescence observation mechanism. 4. The micro force measuring apparatus according to claim 3, wherein the total reflection fluorescence observation mechanism irradiates the observation target surface with an excitation light laser from an objective lens. The probe includes a probe for fixing the fine particles, a light irradiation unit connected to the probe and irradiated with the laser for light pressure, and a support unit for supporting the light irradiation unit. The micro force measuring device according to any one of claims 1 to 4 . The micro force measuring apparatus according to claim 5, wherein the light irradiating unit is made of sheet glass. The micro force measuring device according to claim 5, wherein the light irradiating unit is constituted by a prism. The micro force measuring device according to any one of claims 5 to 7, wherein the base material is fixed to an observation object surface, and the support portion is configured to be movable in a horizontal direction with respect to the observation object surface. 9. The micro force measurement according to claim 8, wherein the light irradiation part and the support part are formed into an integral plate shape, and the integral light irradiation part and the support part are installed in a direction perpendicular to the surface of the object to be observed. apparatus. The micro force measuring device according to claim 8, wherein the support portion is further configured to be movable in a direction perpendicular to an observation object surface. 11. The micro force measuring apparatus according to claim 1, wherein a laser radiation pressure is applied by irradiating the laser for light pressure from an objective lens. The probe position control mechanism is
A position detecting light irradiating means for irradiating the probe with light;
A projecting means for projecting the interference image by the light irradiation onto the split photodiode;
Detection means for detecting and measuring the position of the probe from the interference image;
The micro force measuring device according to claim 1, further comprising an adjusting unit that adjusts the intensity of the laser for light pressure applied to the probe based on a detection result by the detecting unit. The probe position control mechanism monitors and controls that the fluctuation of the probe is converted to a mean square displacement and is held at 0.6 nanometers or less, so that the fluctuation of the probe due to thermal fluctuation or the displacement of the probe due to external force can be reduced. The micro force measuring device according to claim 1, which is configured to prevent the micro force. An offset function is added so that a probe having a constant spring constant is connected to a fixture, and a radiation pressure of an arbitrary intensity is applied to the probe by an optical pressure laser so that dynamic equilibrium can be maintained. The micro force measuring device according to any one of 13.

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