Patent Application
20230303961
The present invention relates to a sample measurement apparatus, a sample measurement system, and an artificial insemination apparatus.
As research and development of new materials for electronics to which regenerative medicine and biological functions are applied become active, visualization of biological material functions is a problem. That is, in a case of a cell, a process monitor of differentiation and induction and a technique of quantitatively measuring signal propagation between an ion channel and an organelle are required.
Meter measurements of processes and functions illustrated here should be considered as various physical quantities such as an electric field, a magnetic field, a temperature, pH, an ion flow, and light emission accompanying a reaction. In this regard, techniques are widely used to, for example, trace a reaction process by using a fluorescent dye that is likely to accompany a specific protein as a label, or to trace the reaction process by extracting a specific organelle in a cell by a centrifugal separation technique or the like, and then analyzing genes. However, there are problems with spatial resolution being at a submicrometer level, which is defined by a wavelength of light, and high degree of invasiveness, such as destruction of the cell.
In recent years, a diamond material including an NV center (nitrogen-vacancy pair) has attracted attention for such a biofunctional measurement because the diamond material has high sensitivity to a minute electromagnetic field and a temperature, has high spatial resolution because a size of a sensor is at an atomic level, and is excellent in biocompatibility because the material is a carbon material.
A measurement technique using the NV center includes (1) a method of using a nanoparticle including the NV center and (2) a method of a scanning probe microscope using a diamond probe including the NV center (for example, see JP2015-529328A (Japanese Patent No. 6117926)).
However, in the measurement using the nanoparticle including the NV center in the method (1), only a vicinity of a portion where the nanoparticle is present can be measured, and position controllability is not high. When the nanoparticles are aggregated, there is a problem that the spatial resolution deteriorates accordingly.
In the above method (2) of the scanning probe microscope using the probe including the NV center, high spatial resolution can be expected, and there is no problem with physical quantities, such as a magnetic field and an electric field, that can be measured in a non-contact manner with respect to a sample.
However, in JP2015-529328A (Japanese Patent No. 6117926), since there is no mechanism that applies an external stimulus to a cell, it is not possible to evaluate cell changes that is dynamics such as before and after insemination, before and after drug stimulation, and before and after neural firing signal input.
In a case of an apparatus configuration illustrated in JP2015-529328A (Japanese Patent No. 6117926), that is, in the case of the configuration in which a laser light that excites the NV center is emitted from the probe side when the sample is scanned using a probe, there is a problem that a space between a lens and the sample can be usually secured only by several mm due to a characteristic of the lens that converges the laser light, and it is difficult to provide the above mechanism that applies the external stimulus.
An object of the invention is to make it possible to provide a mechanism that applies an external stimulus and to measure, by an NV center, structural and electromagnetic changes of a cell due to the external stimulus with high sensitivity.
A sample measurement apparatus according to an aspect of the invention measures a state of a sample using a probe made of diamond or silicon carbide including a nitrogen-vacancy pair. The sample measurement apparatus includes an environment control mechanism provided in a vicinity of the sample, and the environmental control mechanism changes the state of the sample by applying an external stimulus to the sample.
An artificial insemination apparatus according to an aspect of the invention measures a reaction of a cell using a probe made of diamond or silicon carbide including a nitrogen-vacancy pair. The artificial insemination apparatus includes an environment control mechanism provided in the vicinity of the sample, and the environment control mechanism changes a state of the cell by applying the external stimulus to the cell.
According to an aspect of the invention, a mechanism that applies an external stimulus can be provided, and structural and electromagnetic changes of a cell due to the external stimulus can be measured with high sensitivity by an NV center.
Hereinafter, embodiments of the invention will be described in detail with reference to drawings. In all the drawings illustrating the embodiments, the same components are denoted by the same reference numerals in principle, and the repeated description thereof is omitted.
First, a general technique serving as a premise of the invention will be described with reference to
First, a unit cell structure of diamond including an NV center is illustrated in
Generally, a constant number of vacancies (V) is introduced into a diamond substrate containing nitrogen (N) by electron beam irradiation or the like. Then, by performing high-temperature annealing, nitrogen (N) and vacancies (V) are rearranged at positions adjacent to each other in a <111> direction, and are stabilized in terms of energy. A luminescent center formed in the diamond is called an NV center due to an atomic structure thereof.
Such a crystal takes an electron energy state having characteristics illustrated in
In this case, the red fluorescence decreases accordingly. The state of ms=±1 is degenerate in the absence of a magnetic field, but splits into two levels due to Zeeman splitting in the presence of the magnetic field. By taking advantage of this feature, it is possible to accurately measure a resonance level by electron spin resonance (ESR) by sweeping the wavelength of the microwave that excites the electron from the state of ms=0 to the state of ms=±1. A Zeeman splitting width is proportional to the magnetic field sensed by the NV center, and the magnetic field can be measured based on the above two-level width.
That is, when the magnetic field is applied to the NV center, a resonance spectrum on a right side in
It is known that in this ODMR spectrum, an energy (frequency) shift further occurs when there is a temperature change at an NV center position, and as illustrated in
A measurement technique using the NV center may have an atomic level spatial resolution because a sensing portion thereof has an atomic level size as illustrated in
Regarding the method (1) using the nanoparticle including the NV center, a carbon nanoparticle including the NV center is already commercially available and is contained in the sample to be measured. The red fluorescence illustrated in
Next, an outline of an apparatus configuration of the scanning probe microscope using the diamond probe including the NV center in the method (2) will be described. The apparatus configuration will be described based on a general scanning probe microscope (SPM).
That is, a sample is placed on a sample stage on an optical bench having a vibration isolation function, and the sample is scanned from above by the diamond probe including the NV center. A microwave antenna applied to the sample is provided in a vicinity of the sample.
On the other hand, emitted light from the green laser light having the wavelength of 532 nm that excites the electron is aligned, and the green laser light is transmitted through a transparent base material, and the probe is irradiated with the green laser light. During this process, the green laser light passes through an acousto-optic modulator (AOM). The AOM forms a stationary wave in the crystal by a vibration of a piezoelectric element, and uses the stationary wave as a diffraction grating. Since a grating width of the diffraction grating can be controlled by a vibration frequency applied to the crystal, an angle of the light bent through the diffraction grating can be freely changed. Here, instead of the above AOM, another modulator such as an electro-optic modulator (EOM) may be applied.
The NV center is provided at a tip of the probe, and the red fluorescence from the NV center passes through an optical path opposite to that of an incident laser light and is detected by an avalanche photodiode detector (APD detector) via a half mirror. The green laser light is guided to a beam profiler in order to adjust a beam intensity and a shape, and the red fluorescence is guided to a spectrometer in order to grasp a light emission characteristic of the red fluorescence.
A microwave power amplifier and the APD detector are connected to a control system device, and the probe microscope is connected to an SPM controller. Both of these devices are connected by communication, and are mechanisms that can control time sequences of microwave, sample stage control, and the like. These devices are provided in a dark room in order to avoid stray light from entering the detector.
As an electromagnetic field measurement method, high spatial resolution measurement of about several tens of nanometers is expected. The diamond probe including the NV center is formed of diamond together with the transparent base material.
In the measurement using the nanoparticle including the NV center in the method (1), only a vicinity of a portion where the nanoparticle is present can be measured, and position controllability is not high. When the nanoparticles are aggregated, there is a problem that the spatial resolution deteriorates accordingly. When the nanoparticle is introduced into a cell, it is extremely difficult to discharge the nanoparticle to an outside of the cell after evaluation and measurement, and thus it is difficult to use the cell selected for good or bad for artificial insemination or regenerative medicine.
In the method (2) of the scanning probe microscope using the probe including the NV center, the high spatial resolution can be expected, and there is no problem with physical quantities, such as a magnetic field and an electric field, that can be measured in a non-contact manner with respect to the sample.
As described above, in a microscope system using a diamond probe including a general NV center, it is possible to observe an electromagnetic field with high resolution by bringing the NV diamond probe into contact with the sample. However, no mechanism is provided to control an environment of the sample, and it is not possible to evaluate the state change of the sample during drug stimulation and insemination.
since it is necessary to bring a lens having a high light collection rate close to the sample in order to execute high-sensitivity fluorescence detection, a space around the sample is narrow, and it is difficult to provide a mechanism to control the environment.
As described above, since there is no mechanism that applies an external stimulus to a cell, it is not possible to evaluate cell changes that is dynamics such as before and after insemination, before and after drug stimulation, and before and after neural firing signal input.
That is, in a case of a configuration in which a laser light that excites the NV center is emitted from the probe side when the sample is scanned using a probe, there is a problem that a space between the lens and the sample can be usually secured only by several mm due to a characteristic of the lens that converges the laser light, and it is difficult to provide the above mechanism that applies the external stimulus.
According to the invention, a mechanism that applies external stimulus can be provided, and structural and electromagnetic changes of a cell due to the external stimulus can be measured with high sensitivity by the NV center.
Therefore, in the invention, an environment control mechanism that changes the state of the sample is provided in the vicinity of the sample. A space around a sample chamber is secured by providing a mechanism related to the laser light that excites the NV center or the microwave, the probe, and the environment control mechanism on opposite surfaces to each other with respect to the sample stage, and the environmental control mechanism having a wider range of applications can be provided.
Hereinafter, embodiments of the invention will be described with reference to the drawings. Embodiments of the invention relate to a probing apparatus that measures an electromagnetic field, a temperature, a pH value, a composition, and spin information of a soft material in a liquid such as a cell with high spatial resolution.
A sample measurement apparatus according to a first embodiment of the invention will be described with reference to
Here,
In this apparatus configuration, a sample 1 is placed in a culture solution 2 on a sample stage 3. The entire apparatus to be described below is assembled on a vibration isolation table 4 in order to achieve measurement with high spatial resolution, and vibrations of members are prevented as much as possible. A diamond probe 10 including an NV center 11 formed at a tip of the diamond probe 10 is fixed on a probe base 9 made of glass or metal. The diamond probe 10 and the probe base 9 are fixed on a crystal oscillator 8 on a piezoelectric element 7.
Positions of the diamond probe 10 and the probe base 9 are controlled by a probe microscope control mechanism 25, and the diamond probe 10 is brought into contact with the sample 1. Although diamond is used as a material here, a similar quantum sensor material with a defect made of silicon carbide as a light emission point has also attracted attention, and it is possible to replace the probes made of these materials depending on applications.
In the measurement using the NV center 11 of the diamond, fluorescence is excited by the green laser light having the wavelength of 532 nm. Laser light 13 emitted from a laser light source 12 is emitted onto the sample 1 by a mirror 14 and a lens 5. As illustrated in
In the former case, a fluorescence amount is detected as a current or the number of fluorescence pulses, and in the latter case, a fluorescence image on a surface of the sample 1 is captured. Here, in order to align a positional relation between the lens 5 and the laser light, a lens fine adjustment mechanism 19 using the piezoelectric element, a motor, or the like is used. The laser light achieves various quantum measurement protocols by a time sequence in which a microwave and a timing to be described below are matched.
Therefore, in order to form a short time pulse with a high time resolution, an acousto-optic modulator 21, that is, a device called the acousto-optic modulator (AOM) is widely used between the laser light source 12 and the sample 1. A laser light intensity and the like are adjusted by a laser controller 27. A detector controller 22 and a CCD camera controller 23 are used for operation control and signal processing of the detector 16 and the CCD camera 17, respectively.
Next, the microwave, which is another irradiation element, will be described. As described with reference to
A relative positional relation between the sample stage 3 and the diamond probe 10 is controlled by a sample stage fine movement mechanism 20. The sample stage fine movement mechanism 20 is a mechanism capable of coarse movement and fine movement in x, y, and z directions of the sample stage 3 illustrated in the drawings, and a drive source controlled by a control mechanism for the sample stage fine movement controller 26 is formed using a conductive motor and a piezoelectric element in combination.
In order to control an electron spin of the NV center 11, it is necessary to apply a stable external magnetic field. Here, a pair of air core Helmholtz coils 31 sandwich the sample 1, and an intensity and a timing of a magnetic field generated by a Helmholtz coil controller 24 are controlled. The entire system described above is controlled by a system control PC 30.
As illustrated in
First, as illustrated in
Next, for example, in order to fix the cell, it is effective to make the sample suction tube 40 to a slightly negative pressure, and the sample suction tube position and pressure control mechanism 42 controlled by a sample suction controller 43 is also used to adjust the pressure in the sample suction tube 40 for suctioning, fixing, and releasing the cell.
Next, the injection tube 41 is provided in a form of being inserted into the sample 1. In an evaluation of a process of the artificial insemination, in an evaluation of the effects of a sperm and gene editing, in an evaluation of drug effects in a gene drug such as CRISPR-Cas9 and a drug discovery research, and in an evaluation of a drug solution, a neuronal firing mechanism and a cell behavior, an ionic solution such as calcium or potassium is injected into a target organelle inside or outside the cell. For this, a glass pipetle-shaped member having a tip diameter at a micrometer level is assumed. An injection tube position control mechanism 44 formed by the motor and the piezoelectric element is used to cause the injection tube 41 to approach and to be inserted into the target cell. An injection controller 45 is also used to control the amount of injection drug here.
A large number of biological samples are fairly sensitive to changes in states depending on an environmental temperature. Therefore, as the environment control mechanism, temperature adjustment of the sample 1 and flow rate control of the culture solution 2 are important. Here, a heater 46 whose temperature is adjusted by a heater adjustment controller 47 is embedded in the sample stage 3.
A flow path 48 is provided in the sample stage 3, and the culture solution 2 flows by a pump 49 controlled by a flow rate controller 50. Various controllers illustrated in this figure are controlled by a system control PC 30.
Since a large number of mechanisms approach the sample 1, it is necessary to arrange the mechanisms such that the mechanisms do not interfere with one another. That is, in
In particular, since it is essential for efficient microwave irradiation to bring the microwave antenna 18 close to the NV center 11 present in the diamond probe 10 by a distance of several tens of microns, it is effective to bring the microwave antenna 18 close to the NV center 11 from an open side of the diamond probe 10.
On the other hand, since the sample 1 is in the culture solution 2, for a high-precision measurement, it is effective to perform measurement with the diamond probe 10 while fixing the sample 1 with the sample suction tube 40 and injecting the sperm or drug into or out of the cell with the injection tube 41.
Since the injection tube 41 and the diamond probe 10 particularly exert a force when inserted into the sample 1, it is effective for stably fixing the sample 1 to provide the sample suction tube 40 at a position facing the injection tube 41 and the diamond probe 10.
The rotary table 76 in this figure has an independent double rotary stage structure, and is provided on the vibration isolation table 4 in a form of surrounding the sample stage 3. In the example in this figure, a side structure 6 that supports the sample suction tube 40 and a side structure 6 that supports the microwave antenna 18 are placed on the rotary stage in an inner circumference. A side structure 6 that supports the injection tube 41 is placed on the rotary stage on an outer circumference. A side structure 6 that supports the diamond probe 10 is placed on the vibration isolation table 4.
The relative angle relation between the mechanisms can be freely set by a combination of the angles of the rotary stage on the inner and outer circumferences. In particular, in a case in which the diamond probe 10 needs to be inserted into the sample 1 with a strong force, the suction performed by the sample suction tube 40 needs to be strong, and in this case, it is effective that the diamond probe 10 and the sample suction tube 40 face each other with the sample 1 interposed therebetween. Therefore, by the rotary table 76, for example, it is possible to change the disposition such that the sample suction tube 40 can approach the sample 1 from the left, the microwave antenna 18 can approach the sample 1 from below, and the injection tube 41 can approach the sample 1 from above.
The rotary table 76 may not have a double structure as illustrated in
As described above, in the first embodiment, the environment control mechanism is provided in the vicinity of the sample 1 in the sample measurement apparatus that measures the state of the sample 1 using the probe made of diamond or silicon carbide including the nitrogen-vacancy pair (NV center 11). The environment control mechanism changes the state of the sample 1 by applying the external stimulus to the sample 1.
In the first embodiment, an environment control mechanism is provided in the vicinity of the sample 1 in an artificial insemination apparatus that measures a reaction of a cell using the probe made of diamond or silicon carbide including the nitrogen-vacancy pair (NV center 11). The environment control mechanism changes the state of the cell by applying the external stimulus to the cell.
The environment control mechanism includes the sample suction tube 40 that determines a relative positional relation between the sample 1 floating in a solution (culture solution 2) and the probe and that fixes the sample 1 such that the sample 1 does not move in the solution.
The environment control mechanism includes the injection tube 41 that is inserted into the sample 1 and that injects a substance into the sample.
The environment control mechanism includes the sample suction tube 40 that determines the relative positional relation between the sample 1 floating in the solution and the probe and that fixes the sample 1 such that the sample 1 does not move in the solution, the injection tube 41 that is inserted into the sample and that injects a substance into the sample. The sample suction tube 40 and the injection tube 41 face each other at a predetermined angle with respect to the nitrogen-vacancy pair.
The environment control mechanism includes the heater 46 that is disposed inside the sample stage 3 and that adjusts the temperature of the sample, the flow path 48 that is disposed inside the sample stage 3 and that controls the flow rate of the solution.
According to the first embodiment, it is possible to cause a cell culture environment to be constant, reduce the deterioration of the culture solution, and execute evaluation measurement that is more stable for a longer period of time and that is close to an environment in an animal body.
A sample measurement apparatus according to a second embodiment of the invention will be described with reference to
As illustrated in
In the second embodiment, the microwave antenna 18 and the laser light source 12 are provided below the sample stage 3, and can approach the sample 1 from a surface opposite to the diamond probe 10 via the sample 1. Accordingly, a space between the sample and the lens 5 on the diamond probe 10 side is free, the sample environment control mechanism (see
The NV fluorescence excited by the sample 1 is emitted downward from a bottom surface of the sample stage 3 again, and is measured by the detector 16 provided below the sample stage 3. Therefore, the lens 5 is provided below the sample. In the second embodiment, the microwave antenna 18 is also provided under the bottom surface of the sample stage 3.
Since a distance between the microwave antenna 18 and the sample 1 is generally required to be about several tens of micrometers, the bottom surface of the sample stage 3 is assumed to be a thin quartz glass plate or the like. Since the microwave around 2.87 GHz used in the measurement of NV diamond is strongly absorbed in the solution, this method in which the microwave antenna 18 is not inserted into the culture solution has a great advantage.
In the second embodiment, an imaging system used for rough observation such as visual field search is added in an initial stage of observation in a wide visual field from an upper surface of the sample 1. That is, the sample 1 is irradiated with illumination light 61 emitted from a light source 60 such as an LED through the lens 5. Fluorescence 64 generated in the sample 1 is captured by the CCD camera 17.
Here, imaging different from that of the CCD camera 17 illustrated in the first embodiment is considered. That is, an LED light source that is less expensive than a laser is used as the light source 60, and when the green light is illuminated as in the first embodiment, only NV diamond forms a red fluorescent image. However, the light source is changed to blue light, so that NV diamond does not emit light, and an organic substance such as a cell emits the green light. An optical microscope image called a normal bright field image can be obtained by emitting white light.
In this way, by providing the irradiation light to the sample 1 in a system different from that of the laser light, it is possible to provide visual field identifying observation suitable for a structure and a composition of the sample. Accordingly, position identifying accuracy of the NV diamond is improved, and an evaluation in which identification accuracy of a cell structure is improved is achieved. This is an effect of effectively utilizing a space created by causing the apparatus configuration to be the inverted configuration.
Since the inverted configuration is a structure in which light is emitted from above and below the sample 1, the inverted configuration is particularly effective in a case of an optically transparent biological sample or an organic sample. On the other hand, it is not suitable for an evaluation of a sample having a low light transmittance such as a metal material sample or a semiconductor sample, and the configuration according to the first embodiment is effective for such a sample 1.
A third embodiment of the invention will be described with reference to
In the third embodiment, a microsampling method using a focused ion beam processing apparatus (hereinafter referred to as FIB) will be described with reference to
This processing method itself is an example of a general FIB microsampling method, and for details, refer to literatures (for example, T. Ishitani, H. Tsuboi, T. Yaguchi and H. Koike, J. Electron Microsc 43 (1994) pp. 322 to 326).
First, a region of the substrate to be used as a diamond probe later is set as a microsample region 70, a groove is formed around the microsample region 70 with an ion beam, and a microprobe 71 is brought into contact with the microsample region 70. Next, an organic tungsten gas or a phenanthrenegas flows in the FIB apparatus, and a tip of the microprobe 71 is irradiated with an ion beam or an electron beam to solidify the organic tungsten gas or the phenanthrenegas to form an adhesive, thereby fixing the microsample region 70 and the microprobe 71 (
Next, the microsample region 70 is extracted by the microprobe 71 and moved to a base material 72 (
In this way, a needle-shaped diamond probe 75 having a desired shape, for example, a diameter of 1 μm or less and a length of 20 μm or more is formed by FIB processing from various directions illustrated in
Electron micrographs of a tip of the diamond probe 75 actually fabricated from the diamond substrate are illustrated in
In the FIB processing illustrated in
It is confirmed that, by processing the probe to be thinner in the FIB processing illustrated in
As described above, in the third embodiment, the probe has a length of 20 μm or more in the axial direction.
The probe has an axis perpendicular radius of 1 μm or less.
The axis perpendicular radius of the probe at a position of 100 nm from the tip of the probe is equal to or less than a half of the axis perpendicular radius at a position of 1 μm from the tip of the probe.
The above embodiment includes a probe made of diamond including a nitrogen-vacancy pair or silicon carbide including a silicon point defect as a base material, a microwave application mechanism, a laser light source, a photodetector, and a sample environment control mechanism.
A microscope apparatus includes a sample stage capable of holding a liquid sample, and a control mechanism that controls the laser light source and the microwave application mechanism, that analyzes a signal from the photodetector, and that displays a result of the analysis. The microscope apparatus further includes a mechanism that fixes of the sample in the sample stage and controls a position of the sample, and a substance control mechanism that can be inserted into the sample and inject or suction a substance into or from a cell.
The probe, the mechanism that fixes the sample and controls the position of the sample, and the substance control mechanism are disposed on an open surface side of the sample stage. The sample is irradiated with the laser light from a bottom surface side of the sample stage, and the obtained fluorescence is detected from the bottom surface side. The sample environment controller controls a sample temperature control mechanism, a flow rate of a solvent in the sample stage, and an additive.
According to the above embodiments, a mechanism that applies an external stimulus can be provided, and structural and electromagnetic changes of a cell due to the external stimulus can be measured with high sensitivity by the NV center.
As described above, the invention made by the inventor is specifically described based on the embodiments. However, the invention is not limited to the embodiments described above, and various modifications can be made without departing from the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-046774 | Mar 2022 | JP | national |
