Sample measuring device, sample measuring system, and artificial insemination device

Abstract

To allow a mechanism for imparting an external stimulus to be installed so as to enable high-sensitivity measurement of structural and electromagnetic changes of a cell in response to the external stimulus using an NV center.SOLUTION: A sample measurement device provided herein has an environmental control mechanism for changing the state of a sample by imparting an external stimulus to the sample.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a sample measuring device, a sample measuring system, and an artificial insemination device. [Background technology]

[0002] As research and development of new materials for electronics that utilize regenerative medicine and biological functions becomes more active, visualizing the functions of biomaterials is a challenge. Specifically, in the case of cells, technologies are needed to monitor the differentiation and induction processes, and to quantitatively measure signal propagation between ion channels and organelles.

[0003] The processes and functions described here should be understood as various physical quantities such as electric field, magnetic field, temperature, pH, ion flow, and luminescence associated with reactions. In this context, techniques such as using fluorescent dyes that tend to associate with specific proteins as labels to track reaction processes, or extracting specific organelles within cells using centrifugation techniques and tracking reaction processes through gene analysis, are widely used. However, these techniques have problems such as having a spatial resolution at the submicrometer level, defined by the wavelength of light, and being highly invasive, such as destroying cells.

[0004] In recent years, diamond materials containing NV centers (nitrogen-vacancy pairs) have attracted attention for their high sensitivity to minute electromagnetic fields and temperature, their high spatial resolution due to their atomic-level sensor size, and their excellent biocompatibility due to their carbon material composition, making them ideal for measuring biological functions.

[0005] Measurement techniques using NV centers include (1) a method using nanoparticles containing NV centers, and (2) a scanning probe microscope method using a diamond probe containing NV centers (see, for example, Patent Document 1).

[0006] However, in measurements using nanoparticles containing NV centers as described in (1), only the area surrounding the nanoparticles can be measured, and the positional controllability is not high. Furthermore, if the nanoparticles are aggregated, there is a problem in that the spatial resolution deteriorates accordingly. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Special Publication No. 2015-529328 (Patent No. 6117926) [Overview of the project] [Problems that the invention aims to solve]

[0008] In scanning probe microscopy using a probe containing an NV center as described in (2) above, high spatial resolution can be expected, and there are no problems with measuring physical quantities that can be measured non-contact with the sample, such as magnetic fields and electric fields.

[0009] However, since Patent Document 1 does not involve a mechanism for applying external stimuli to cells, it is not possible to evaluate cellular changes, i.e., dynamics such as before and after fertilization, before and after drug stimulation, and before and after the input of nerve firing signals.

[0010] Furthermore, in the apparatus configuration shown in Patent Document 1, that is, in which a laser beam is irradiated from the probe side to excite the NV center when the probe is scanned over the sample, due to the characteristics of the lens that focuses the laser beam, the space between the lens and the sample can usually only be a few millimeters, which presents the challenge of making it difficult to install the mechanism that provides the external stimulus mentioned above.

[0011] The objective of the present invention is to enable the installation of a mechanism for applying external stimuli, thereby allowing for highly sensitive measurement of structural and electromagnetic changes in cells in response to external stimuli using an NV center. [Means for solving the problem]

[0012] A sample measuring device according to one aspect of the present invention is a sample measuring device that measures the state of a sample using a probe made of diamond or silicon carbide containing nitrogen-vacancy pairs, and is characterized in that it has an environmental control mechanism installed near the sample, and the environmental control mechanism changes the state of the sample by applying an external stimulus to the sample.

[0013] An artificial insemination apparatus according to one aspect of the present invention is an artificial insemination apparatus for measuring the response of cells using a probe made of diamond or silicon carbide containing nitrogen-vacancy pairs, and is characterized in that it has an environmental control mechanism installed near the sample, and the environmental control mechanism changes the state of the cells by applying an external stimulus to the cells. [Effects of the Invention]

[0014] According to one aspect of the present invention, a mechanism for applying external stimuli can be installed, enabling highly sensitive measurement of structural and electromagnetic changes in cells in response to external stimuli using an NV center. [Brief explanation of the drawing]

[0015] [Figure 1A] This figure shows the crystal structure of diamond containing NV centers. [Figure 1B] This figure shows the optically detected magnetic resonance spectrum. [Figure 1C] This is a diagram showing the state of electron energy. [Figure 2] This is a front view showing the basic configuration of an upright probe device using a diamond with an NV center as a probe in Example 1. [Figure 3] This is a side view showing the basic configuration of an upright probe device using a diamond with an NV center as a probe in Example 1. [Figure 4] This is a front view showing the basic configuration of an inverted probe device using a diamond with an NV center as a probe in Example 2. [Figure 5]This is a diagram showing the manufacturing process of a probe for a probe device by a convergent ion beam and a micro-sampling method in Example 3. [Figure 6] This is a top view showing the basic configuration of a probe device using a diamond having an NV center in Example 1 as a probe. [Figure 7] This is a top view showing the basic configuration of a probe device having a turntable using a diamond having an NV center in Example 1 as a probe.

Embodiments for Carrying Out the Invention

[0016] Hereinafter, embodiments of the present invention will be described in detail based on the drawings. In all the drawings for explaining the embodiments, the same members are basically denoted by the same reference numerals, and the repeated explanations thereof are omitted.

[0017] First, referring to FIGS. 1A, 1B, and 1C, general techniques that are the premise of the present invention will be described.

[0018] First, the unit cell structure of a diamond having an NV center is shown in FIG. 1A.

[0019] Generally, a certain amount of vacancies (V) is introduced into a diamond substrate containing nitrogen (N) by electron beam irradiation or the like. After that, by performing high-temperature annealing, nitrogen (N) and vacancies (V) are rearranged to adjacent positions in the <111> direction and are stabilized energetically. This luminescence center formed in the diamond is called an NV center from its atomic structure.

[0020] Such crystals will take on the characteristic electron energy states shown in FIG. 1C. The NV pair usually captures one electron to become a monovalent NV - and the electrons form a spin triplet state. When irradiated with green light having a wavelength of 532 nm in a state where nothing is done, the electrons excited from the m s = 0 state emit red fluorescence with a longer wavelength (about 550 to 800 nm) to return to the original m sRelaxes to the state of =0. On the other hand, when this crystal is irradiated with microwaves near 2.87 GHz, electrons are excited from the m s =0 state to the m s =±1 state by electron spin resonance. When the m s =±1 state is irradiated with the above-mentioned green light with a wavelength of 532 nm, a part of the electrons relaxes to the m s =0 state via non-radiative transition.

[0021] In this case, the red fluorescence will decrease accordingly. The m s =±1 states are degenerate in the absence of a magnetic field, but when there is a magnetic field, Zeeman splitting occurs and they split into two levels. Taking advantage of this feature, by sweeping the wavelength of the microwave that excites electrons from the m s =0 state to the m s =±1 state, it becomes possible to accurately measure the resonance levels by electron spin resonance (ESR: Electron Spin Resonance). The Zeeman splitting width is proportional to the magnetic field felt by the NV center, and the magnetic field can be measured from the above-mentioned two-level width.

[0022] That is, when a magnetic field is applied to the NV center, the resonance spectrum on the right in Fig. 1C is obtained, and the applied magnetic field can be calculated from the energy difference (here, the frequency difference) between the two peaks. For example, when a magnetic field of 1 gauss (0.1 mT) is applied to the NV center at room temperature, it is known that the peak interval is about 2.8 MHz apart. This spectrum is generally called an optically detected magnetic resonance (ODMR) spectrum.

[0023] It is known that when there is a temperature change at the NV center position, further energy (frequency) shift occurs in this ODMR spectrum. As shown in Fig. 1B, at room temperature of 300 K, a shift amount of -75 kHz / K and at 500 K, a shift amount of -140 kHz / K are obtained. Therefore, the temperature at which the NV center is located can be measured from this shift amount, and its accuracy is said to be 10 mK or less.

[0024] Measurement techniques utilizing NV centers have the potential to possess atomic-level spatial resolution because their sensing portion is atomic-level in size, as shown in Figure 1A. However, because detection is based on light, the emission point is spread to the wavelength of light, i.e., several hundred nanometers. Therefore, to achieve high spatial resolution when measuring the electromagnetic field and temperature of a sample with NV centers, there are two approaches: (1) using nanoparticles containing NV centers, and (2) using a scanning probe microscope with a diamond probe containing NV centers.

[0025] Regarding the method using nanoparticles containing NV centers described in (1), carbon nanoparticles containing NV centers are already commercially available, and this method involves embedding them in the sample to be measured. By irradiating these with a 532 nm green laser beam and microwaves, the red fluorescence shown in Figures 1B and 1C is detected. The spatial resolution is obtained by the nanoparticle size if the position of the nanoparticles can be accurately recognized. Here, the green laser beam described above can be said to be optimally excited if its wavelength is between 520 nm and 540 nm. On the other hand, it is also possible to use a yellow-green laser beam with a wavelength of 561 nm, for example, as the laser beam used for irradiation.

[0026] Next, we will describe the overview of the system configuration of a scanning probe microscope using a diamond probe including the NV center described in (2). Here, we will describe the system configuration based on a general scanning probe microscope (SPM).

[0027] Specifically, the sample is placed on a sample stage on an optical table with vibration isolation capabilities, and is scanned from above with a diamond probe including an NV center. A microwave antenna for applying microwaves to the sample is installed near the sample.

[0028] On the other hand, the emission from a 532nm wavelength green laser light that excites electrons is aligned, transmitted through a transparent substrate, and irradiated onto the probe. Along the way, the green laser light passes through an AOM (Acousto-Optic Modulator). An AOM creates a standing wave in a crystal by vibration using a piezoelectric element and uses this as a diffraction grating. Since the grating width of the diffraction grating can be controlled by the vibration frequency applied to the crystal, the angle at which light bends through the diffraction grating can be freely changed. Here, it is also possible to apply other modulators such as an EOM (Electro-Optic Modulator) instead of the AOM mentioned above.

[0029] The probe tip has an NV center, from which red fluorescence travels along the opposite optical path to the incident laser light, passes through a half-mirror, and is detected by an avalanche photodiode detector (APD detector). The green laser light is guided to a beam profiler to adjust the beam intensity and shape, and the red fluorescence is guided to a spectrometer to understand its emission characteristics.

[0030] The microwave power amplifier and APD detector are connected to the control system, and the probe microscope is connected to the SPM controller. These two systems are also connected via communication, allowing for control of the time sequence, including microwave and sample stage control. These components are installed in a darkroom to avoid stray light entering the detector.

[0031] This electromagnetic field measurement method is expected to enable high spatial resolution measurements of several tens of nanometers. The diamond probe, including the NV center, is made of diamond, along with the transparent substrate.

[0032] In measurements using nanoparticles containing NV centers as described in (1), only the area surrounding the nanoparticles is measurable, resulting in poor positional control. Furthermore, if the nanoparticles are aggregated, the spatial resolution deteriorates, posing a challenge. Moreover, when nanoparticles are introduced into cells, it is extremely difficult to expel the particles after evaluation and measurement, making it difficult to use cells selected for quality in artificial insemination or regenerative medicine.

[0033] Furthermore, scanning probe microscopy using a probe containing an NV center as described in (2) offers high spatial resolution, and there are no issues with measuring physical quantities such as magnetic fields and electric fields without contact with the sample.

[0034] Thus, while microscope systems using diamond probes containing conventional NV centers allowed for high-resolution electromagnetic field observation by contacting the NV diamond probe with the sample, they lacked a mechanism to control the sample environment, making it impossible to evaluate changes in the sample's state during drug use or fertilization.

[0035] Furthermore, because a highly sensitive lens with high light-gathering power had to be placed close to the sample in order to perform high-sensitivity fluorescence detection, the space around the sample was limited, making it difficult to install an environmental control mechanism.

[0036] Thus, because there is no mechanism to provide external stimuli to cells, it is not possible to evaluate cellular changes, i.e., dynamics such as before and after fertilization, before and after drug stimulation, and before and after the introduction of neural firing signals.

[0037] In other words, when scanning the probe over a sample, if the setup involves irradiating the probe with laser light to excite the NV center, the space between the lens and the sample is usually only a few millimeters due to the characteristics of the lens that focuses the laser light. This presents a challenge in that it is difficult to install the external stimulation mechanism described above.

[0038] The present invention aims to enable the installation of a mechanism for applying external stimuli, thereby allowing for highly sensitive measurement of structural and electromagnetic changes in cells in response to external stimuli using an NV center.

[0039] Therefore, in this invention, an environmental control mechanism that changes the state of the sample is installed near the sample. Furthermore, by installing the mechanism related to the laser light or microwave that excites the NV center and the probe and environmental control mechanism on opposite sides of the sample stage, space around the sample chamber is secured, making it possible to install an environmental control mechanism with a wider range of applications.

[0040] The embodiments of the present invention will be described below with reference to the drawings. The embodiments of the present invention relate to a probing device for measuring the electromagnetic field, temperature, pH value, composition, and spin information of liquid soft materials, including cells, with high spatial resolution. [Examples]

[0041] The sample measuring apparatus of Example 1 of the present invention will be described with reference to Figures 2, 3, 6, and 7. The sample measuring apparatus of Example 1 is a probe device that uses a diamond having an NV center as a probe.

[0042] Here, Figure 2 is a front view showing the basic configuration of an upright probe device using a diamond with an NV center as a probe. Figure 3 is a side view showing the basic configuration of an upright probe device using a diamond with an NV center as a probe. Figure 6 is a top view showing the basic configuration of a probe device using a diamond with an NV center as a probe. Figure 7 is a top view showing the basic configuration of a probe device with a rotating table using a diamond with an NV center as a probe.

[0043] Figure 2 shows the basic configuration of a probe device using a diamond with an NV center as the probe. This configuration is called an upright type because the probe and laser beam approach the sample from the same plane.

[0044] In this apparatus configuration, sample 1 is placed in the culture medium 2 on the sample stage 3. The entire apparatus, as described below, is assembled on a vibration isolation table 4 to achieve high spatial resolution measurements, minimizing vibration of the components. The diamond probe 10, with an NV center 11 formed at its tip, is fixed on a probe stand 9 made of glass or metal, and these are fixed on a quartz oscillator 8 on a piezoelectric element 7.

[0045] These probes are positioned by the probe microscope control mechanism 25 and touch the sample 1. Although diamond is used as the material here, similar quantum sensor materials made of silicon carbide, which use defects as light-emitting points, are also attracting attention, and these materials can be used as probes depending on the application.

[0046] In measurements using the diamond NV center 11, fluorescence is excited by green laser light with a wavelength of 532 nm. The laser light 13 emitted from the laser light source 12 is irradiated onto the sample 1 by the mirror 14 and lens 5. As shown in Figure 1, the sample 1 emits red NV fluorescence 15, which is focused by the lens 5 and then focused via the mirror 14 to the detector 16 or the CCD camera 17.

[0047] In the former, the amount of fluorescence is detected as current or the number of fluorescence pulses, while in the latter, a fluorescence image is captured on one surface of the sample. Here, a fine adjustment mechanism 19 for the lens, such as a piezoelectric element or motor, is used to align the positional relationship between the lens 5 and the laser beam. The laser beam is time-sequenced with the microwaves described below to realize various quantum measurement protocols.

[0048] Therefore, in order to form short-time pulses with high temporal resolution, an acousto-optic modulator 21, or AOM (Acousto-Optic Modulator), is widely used between the laser light source 12 and the sample 1. In addition, the laser light intensity and other parameters are adjusted by the laser control mechanism 27. The detector control mechanism 22 and the CCD camera control mechanism 23 are used for the operation control and signal processing of the detector 16 and the CCD camera 17, respectively.

[0049] Next, let's discuss the other irradiation element: microwaves. As shown in Figure 1, the sample needs to be irradiated with microwaves around 2.87 GHz while scanning the frequency. Furthermore, since bringing the microwave source close to the sample 1 at a distance of several tens of micrometers is important for high-sensitivity measurement, a microwave antenna 18 that can be placed close to the sample is installed. The frequency, intensity, timing, etc., of the microwave antenna 18 are controlled by a microwave control mechanism 29.

[0050] The relative positional relationship between the sample stage 3 and the diamond probe 10 is controlled by the sample stage fine-adjustment mechanism 20. This sample stage fine-adjustment mechanism 20 is a mechanism that can coarsely and finely adjust the sample stage 3 in the x, y, and z directions as shown in the figure, and the drive source controlled by the sample stage fine-adjustment mechanism control mechanism 26 is formed by using a combination of a conductive motor and a piezoelectric element.

[0051] To control the electron spin of the NV center 11, a stable external magnetic field must be applied. Here, air-core Helmholtz coils 31 are placed in pairs on either side of the sample 1, and the magnetic field strength and timing generated by the Helmholtz coil control mechanism 24 are controlled. The entire system is controlled by the system control PC 30.

[0052] Figure 3 shows a side view of this upright type device. As shown in Figure 3, a key feature of Example 1 is the inclusion of a sample environment control mechanism.

[0053] First, as shown in Figure 1, sample 1 is suspended in culture medium 2, so the relative positional relationship between sample 1 and the diamond probe 10 cannot be determined by this alone. This is resolved by fixing sample 1 in place in culture medium 2 using the sample suction tube 40. The position of the sample suction tube 40 is controlled by a sample suction tube position and pressure control mechanism 42, which is formed by a motor or piezoelectric element.

[0054] Next, for example, to fix cells, it is effective to create a slight negative pressure in the sample suction tube 40, and the sample suction tube position and pressure control mechanism 42 controlled by the sample suction tube control mechanism 43 is also used to adjust the pressure inside the sample suction tube 40 for aspirating, fixing, and releasing cells.

[0055] Next, an injection tube 41 is positioned in a form that will be inserted into sample 1. In the evaluation of the artificial insemination process, sperm is injected; in the evaluation of the effect of gene editing, gene drugs such as CRISPR-Cas9 are injected; in the evaluation of drug efficacy in drug discovery research, drug solutions are injected; and in the evaluation of the firing mechanism of nerve cells and cell behavior, ionic solutions such as calcium and potassium are injected into the target organelles inside and outside the cells. For this, a glass pipette-shaped component with a tip diameter at the micrometer level is envisioned. An injection tube position control mechanism 44, formed by a motor or piezoelectric element, is used to bring the injection tube 41 close to and insert it into the target cells. An injection tube control mechanism 45 is also used to control the amount of drug injected.

[0056] Many biological samples are highly sensitive to ambient temperature changes. Therefore, environmental control mechanisms such as temperature control of sample 1 and flow rate control of culture medium 2 are crucial. In this configuration, a heater 46, whose temperature is controlled by a heater adjustment mechanism 47, is embedded within the sample stage 3.

[0057] Furthermore, a flow path 48 is provided in the sample stage 3, and the culture medium 2 flows through it via a pump 49 controlled by a flow rate control mechanism 50. The various control mechanisms shown in this figure are controlled by the system control PC 30.

[0058] Figure 6 shows a top view of a probe device using a diamond with an NV center as shown in Figure 2. In particular, this figure depicts the various components installed at the height from the vibration isolation table 4 to the bottom surface of the lens 5, showing the arrangement of each mechanism.

[0059] Since many mechanisms approach sample 1, it is necessary to arrange them so that they do not interfere with each other. Specifically, in Figure 6, with sample 1 at the center, the diamond probe 10 approaches sample 1 from the right, and the microwave antenna 18 approaches sample 1 from the opposite left.

[0060] In particular, since it is essential for efficient microwave irradiation to bring the microwave antenna 18 within a distance of several tens of microns of the NV center 11 located inside the diamond probe 10, it is effective to approach it from the open side of the diamond probe 10.

[0061] On the other hand, since sample 1 is in the culture medium 2, for high-precision measurement, it is effective to fix sample 1 with the sample suction tube 40 while administering sperm or drugs into and out of the cells using the injection tube 41, and then measuring with the diamond probe 10.

[0062] Since the injection tube 41 and diamond probe 10 exert particular force when inserted into the sample 1, positioning the sample suction tube 40 opposite them is effective for stably fixing the sample 1.

[0063] As shown in Figure 6, the sample suction tube 40 and injection tube 41 are positioned opposite each other, approaching the sample 1 from above and below. Similarly, the flow path 48 and pump 49 systems must be arranged so as not to interfere with these mechanisms.

[0064] In Figure 6, the channel 48 is shown as a dotted line, illustrating a configuration in which the channel 48 is drawn out to the right, in the same direction, below the diamond probe 10, quartz oscillator 8, and piezoelectric element 7. The channel 48 can also be rerouted within the sample stage 3, for example, by being drawn out from the lower edge of the sample stage 3 in this figure.

[0065] Figure 7 shows an embodiment in which each mechanism is mounted on a rotating table 76 so that the relative angular relationship of each mechanism that is brought close to the sample 1 can be changed.

[0066] In this figure, the rotating stage 76 has an independent double rotating stage structure, which is installed on the vibration isolation table 4 surrounding the sample stage 3. In this example, the inner rotating stage has a side structure 6 that supports the sample suction tube 40 and another side structure 6 that supports the microwave antenna 18. The outer rotating stage has a side structure 6 that supports the injection tube 41. The side structure 6 that supports the diamond probe 10 is placed on the vibration isolation table 4.

[0067] The relative angular relationships of each mechanism can be freely set by combining the angles of the inner and outer rotating stages. In particular, when the diamond probe 10 needs to be inserted into the sample 1 with strong force, the suction from the sample suction tube 40 must be strong, and in this case, it is effective for both to be positioned facing each other with the sample 1 in between. Therefore, the rotating stage 76 can be used to change the arrangement so that, for example, the sample suction tube 40 can be approached from the left, the microwave antenna 18 from below, and the injection tube 41 from above.

[0068] The rotating platform 76 does not necessarily have to be a double structure as shown in Figure 7, and it is also possible to have a structure that does not have a full rotation stroke, for example, a structure that limits it to half a rotation or less. Furthermore, the side structure 6 can be designed to move in one direction, and each mechanism can be retracted when not in use.

[0069] Thus, in Example 1, a sample measuring device is used to measure the state of a sample 1 using a probe made of diamond or silicon carbide containing nitrogen-vacancy pairs (NV centers 11), and an environmental control mechanism is installed near the sample 1. The environmental control mechanism changes the state of the sample 1 by applying an external stimulus to the sample 1.

[0070] Furthermore, in Example 1, an artificial insemination device was used to measure the cell response using a probe made of diamond or silicon carbide containing nitrogen-vacancy pairs (NV centers 11), and an environmental control mechanism was installed near the sample 1. The environmental control mechanism changes the state of the cells by applying external stimuli to them.

[0071] The environmental control mechanism includes a sample suction tube 40 that determines the relative positional relationship between the sample 1 suspended in the solution (culture medium 2) and the probe, and fixes the sample 1 so that it does not move in the solution.

[0072] Furthermore, the environmental control mechanism is installed to be inserted into the sample 1 and has an injection tube 41 for injecting a substance into the sample.

[0073] Furthermore, the environmental control mechanism includes a sample suction tube 40 that determines the relative positional relationship between the sample 1 suspended in the solution and the probe, and fixes the sample 1 so that it does not move in the solution, and an injection tube 41 that is installed to be inserted into the sample and to inject a substance into the sample. Here, the sample suction tube 40 and the injection tube 41 are arranged to face each other at a predetermined angle with respect to the nitrogen-vacancy pair.

[0074] Furthermore, the environmental control mechanism includes a heater 46 located inside the sample stage 3 for adjusting the temperature of the sample, and a flow path 48 located inside the sample stage 3 for controlling the flow rate of the solution.

[0075] According to Example 1, the cell growth environment can be kept constant, the deterioration of the culture medium can be reduced, and evaluation and measurement can be performed for a longer period of time in a more stable environment that closely resembles the environment inside an animal. [Examples]

[0076] Referring to Figure 4, the sample measuring apparatus of Example 2 of the present invention will be described. The sample measuring apparatus of Example 2 is a probe device that uses a diamond having an NV center as a probe.

[0077] Figure 4 shows a front view of the basic configuration. This configuration is called an inverted type because the probe and laser beam approach the sample from opposite sides.

[0078] As shown in Figure 4, sample 1 is placed in the culture medium 2 within the sample stage 3. The diamond probe 10 is fixed on a probe stand 9 made of glass or metal, and its position is controlled by a probe position control mechanism 62 that has position control in the x, y, and z directions as well as linear motion in the direction of the probe axis. This has the advantage that the diamond probe 10 can be inserted linearly into the sample 1, making it easy to insert the diamond probe 10 without breaking it.

[0079] In this second embodiment, the microwave antenna 18 and laser light source 12 are installed below the sample stage 3, and are characterized by being approached from the opposite side of the sample 1 from the diamond probe 10 via the sample 1. This frees up space between the lens 5 on the diamond probe 10 side, making it easy to install sample environment control mechanisms (see Figure 3) such as the sample suction tube 40, injection tube 41, heater 46, and flow path 48, which are installed on the side view not shown in the figure, and also allows for a larger sample stage 3. In other words, the laser light 13 from the laser light source 12 is irradiated onto the sample 1 from the bottom surface of the sample stage 3. For this reason, the sample stage 3 is made of a material that efficiently transmits laser light, such as a quartz glass petri dish.

[0080] The NV fluorescence excited by sample 1 is again emitted downward from the bottom surface of sample stage 1 and measured by detector 16, which is also installed below it. Therefore, lens 5 is installed below the sample. In this embodiment 2, microwave antenna 18 is also installed below the bottom surface of sample stage 3.

[0081] Since a distance of several tens of micrometers is usually required between the microwave antenna 18 and the sample 1, the bottom surface of the sample stage 3 is assumed to be a thin quartz glass plate or the like. Furthermore, since microwaves around 2.87 GHz used in the measurement of NV diamonds are greatly absorbed in liquid, this method, which does not involve inserting the microwave antenna 18 into the culture medium, has a significant advantage.

[0082] In this second embodiment, an imaging system was added to the top surface of the sample 1 for rough observation, such as finding the field of view, in the initial stages of observation with a wide field of view. Specifically, illumination light 61 emitted from a light source 60 such as an LED is irradiated onto the sample 1 with a lens 5. Fluorescence 64 generated in the sample 1 is captured by a CCD camera 17.

[0083] Here, we consider imaging different from that of the CCD camera 17 shown in Example 1. Specifically, we use an LED light source, which is less expensive than a laser, as the light source 60. When illuminated with green light, as in Example 1, only the NV diamond forms a red fluorescence image. However, by using blue light as the light source, the NV diamond does not emit light, while organic matter such as cells emits light in response to green light. Furthermore, by illuminating with white light, we can obtain an optical microscope image known as a normal bright-field image.

[0084] In this way, by providing a separate light source for irradiating sample 1 from the laser light, it becomes possible to perform field-of-view observations that are suitable for the sample structure and composition. This improves the accuracy of NV diamond positioning and enables evaluation with higher accuracy in identifying cell structures. This is an effect of effectively utilizing the space created by the inverted configuration of the device.

[0085] Furthermore, since the inverted type irradiates light onto sample 1 from both above and below, it is particularly effective for optically transparent biological samples and organic samples. On the other hand, it is not suitable for evaluating samples with low light transmittance, such as metallic materials and semiconductor samples, and the configuration of Example 1 is effective for such samples 1. [Examples]

[0086] Example 3 of the present invention will be described with reference to Figure 5. In Example 3, as an example of processing a probe suitable for a probe, a microsampling method using a Focused Ion Beam (FIB) processing device will be explained with reference to Figure 5.

[0087] This processing method is an example of a general FIB microsampling method; for further details, please refer to the literature (e.g., T. Ishitani, H. Tsuboi, T. Yaguchi and H. Koike, J. Electron Microsc 43 (1994) pp. 322-326).

[0088] First, a region of the substrate that will later become a diamond probe is designated as the microsample region 70, and grooves are machined around it using an ion beam, while the microprobe 71 is brought into contact with the microsample region 70. Next, organic tungsten gas or phenanthrene gas is flowed through the FIB apparatus, and the tip of the microprobe 71 is irradiated with an ion beam or electron beam to solidify the organic tungsten gas or phenanthrene gas, which acts as an adhesive to fix the microsample region 70 and the microprobe 71 (Figure 5(a)).

[0089] Next, the microsample region 70 is extracted with the microprobe 71 and transferred to the substrate 72 (Figure 5(b)). After this, organic tungsten gas or phenanthrene gas is again flowed over the contact area between the substrate 72 and the microsample region 70, and an ion beam is irradiated to form an adhesive layer 74 and fix the two together. Subsequently, the excess sample portion of the microsample region 70 is removed with FIB, and then the fixation of the substrate 72 and the microsample 73 is reinforced to complete the fixation (Figure 5(c)).

[0090] Thus, a needle-shaped diamond probe 75, for example, with a diameter of 1 μm or less and a length of 20 μm or more, is formed on the roughly processed microsample 73 fixed to the substrate 72 by FIB processing from various directions as shown in Figure 5(d).

[0091] Figures 5(f) and 5(g) show electron microscope images of the tip of a diamond probe 75 actually fabricated from a diamond substrate. Figure 5(f) shows a diamond probe using a needle-shaped tungsten substrate, with only the tip being an NV diamond region. Here, the probe diameter is approximately 1 μm and the probe length is approximately 25 μm.

[0092] In the FIB processing shown in Figure 5(d), the tip was processed to have a knife-edge shape. By using this shape-on-probe for the diamond probe 10 in Figure 4, it became possible to insert the diamond probe 10 into the cell with low resistance.

[0093] Furthermore, by further processing this probe to make it thinner in the FIB process shown in Figure 5(d), we confirmed that the probe diameter can be reduced to 200 nm or less, as shown in Figure 5(g). It goes without saying that although we referred to it as a diamond probe above, it is equally possible to apply this method to quantum sensing materials with similar light-emitting centers, such as silicon carbide.

[0094] Thus, in Example 3, the probe has an axial length of 20 μm or more. Furthermore, the probe has an axis perpendicular radius of 1 μm or less. Furthermore, the axial perpendicular radius of the probe at a position 100 nm from the tip is less than or equal to half the axial perpendicular radius at a position 1 μm from the tip of the probe.

[0095] The above embodiment includes a probe made of diamond containing nitrogen-vacancy pairs or silicon carbide having silicon point defects, a microwave application mechanism, a laser light source, a photodetector, and a sample environment control mechanism.

[0096] Furthermore, in a microscope apparatus comprising a sample stage capable of holding liquid samples, a control mechanism that controls a laser light source and a microwave application mechanism, and analyzes signals from a photodetector and displays the results, the apparatus includes a mechanism for fixing and controlling the position of the sample within the sample stage, and a substance control mechanism that can be inserted into the sample and injected or aspirated into cells.

[0097] Furthermore, a mechanism for fixing and controlling the position of the probe and the sample, as well as a substance control mechanism, are arranged on the open side of the sample stage. Laser light is irradiated onto the sample from the bottom side of the sample stage, and the resulting fluorescence is detected from the bottom side. The sample environment control mechanism consists of a sample temperature control mechanism and a mechanism for controlling the flow rate of the solvent and additives in the sample stage.

[0098] According to the above embodiment, a mechanism for applying external stimuli can be installed, enabling highly sensitive measurement of structural and electromagnetic changes in cells in response to external stimuli using an NV center.

[0099] Although the present invention has been specifically described above based on embodiments, it goes without saying that the present invention is not limited to the above embodiments and can be modified in various ways without departing from its essence. [Explanation of Symbols]

[0100] 1: Sample 2:Culture solution 3: Sample stage 4: Vibration isolation table 5: Lens 6: Side structure 7: Piezo element 8: Crystal oscillator 9: Probe base 10: Diamond probe 11: NV Center 12: Laser light source 13: Laser light 14: Mirror 15: NV fluorescence 16: Detector 17: CCD camera 18: Microwave antenna 19: Fine adjustment mechanism for lenses 20: Fine adjustment mechanism for sample stage 21: Acousto-optic modulator 22: Detector control mechanism 23: CCD camera control mechanism 24: Helmholtz coil control mechanism 25: Probe Microscope Control Mechanism 26: Control mechanism for fine adjustment mechanism of sample stage 27: Laser control mechanism 28: Control mechanism for lens fine adjustment mechanism 29: Microwave control mechanism 30: System control PC 31: Helmholtz Coil 40: Sample suction tube 41: Injection tube 42: Sample suction tube position and pressure control mechanism 43: Sample suction tube control mechanism 44: Injection tube position control mechanism 45: Injection tube control mechanism 46: Heater 47: Heater adjustment mechanism 48: Flow Channel 49: Pump 50: Flow control mechanism 60:Light source 61: Illumination light 62: Probe position control mechanism 63: Probe control mechanism 64: Fluorescence 70: Microsample area 71: Microprobe 72: Base material 73: Microsample 74: Adhesive layer 75: Diamond probe 76: Rotating platform

Claims

1. A sample measuring device that measures the state of a sample suspended in a solution using a probe made of diamond or silicon carbide containing nitrogen-vacancy pairs, The system has an environmental control mechanism that changes the state of the sample by applying an external stimulus to the sample, The aforementioned environmental control mechanism is The sample, suspended in the solution, is fixed while remaining suspended in the solution, and the external stimulus is applied to the sample. The aforementioned environmental control mechanism is A sample suction tube is used to determine the relative positional relationship between the sample suspended in the solution and the probe, and to fix the sample in place while it is suspended in the solution so that it does not move. It has an injection tube that is installed to be inserted into the sample and for injecting a substance into the sample, The sample suction tube and the injection tube are, A sample measuring device characterized by being positioned so as to face the nitrogen-vacancy pair at a predetermined angle.

2. It has a sample stand for holding the sample, The aforementioned environmental control mechanism is A heater is placed inside the sample stage and is used to adjust the temperature of the sample. A channel is placed inside the sample stage through which the solution flows, A pump that controls the flow rate of the solution flowing through the aforementioned channel, The sample measuring device according to claim 1, characterized by having the following features.

3. It has a sample stand for holding the sample, The aforementioned environmental control mechanism is The sample measuring device according to claim 1, characterized in that it is arranged in a space formed above the sample stage.

4. The system comprises at least a laser light source for exciting the nitrogen-vacancy pair, a detector, and a first lens. The laser light source, the detector, and the first lens are, The sample measuring device according to claim 3, characterized in that it is positioned below the sample stage.

5. It further has a microwave mechanism, The microwave mechanism described above is The sample measuring device according to claim 4, characterized in that it is positioned below the sample stage.

6. The aforementioned sample stand is It is made of a material that transmits laser light output from the aforementioned laser light source, The sample measuring apparatus according to claim 4, characterized in that the laser light is irradiated onto the sample from the bottom surface of the sample stage.

7. The fluorescence excited by the aforementioned sample is The sample measuring device according to claim 4, characterized in that it emits light downward from the bottom surface of the sample stage and is measured by the detector.

8. The microwave mechanism described above is The sample measuring device according to claim 5, characterized in that it is arranged so as not to be inserted into the solution.

9. Above the aforementioned sample stage, A light source, a second lens, and a camera are arranged. The illumination light emitted from the light source is irradiated onto the sample through the second lens. The sample measuring apparatus according to claim 3, characterized in that fluorescence generated in the sample is captured by the camera.

10. The aforementioned probe is The sample measuring device according to claim 1, characterized in that the axial length is 20 μm or more.

11. The aforementioned probe is The sample measuring device according to claim 1, characterized in that the axial perpendicular radius is 1 μm or less.

12. The aforementioned probe is The sample measuring device according to claim 1, characterized in that the axial perpendicular radius at a position 100 nm from the tip is less than or equal to half the axial perpendicular radius at a position 1 μm from the tip of the probe.

13. A sample measurement system characterized by having a control mechanism that analyzes the measurement signal output from the sample measurement device described in claim 1 and displays the analysis result.

14. An artificial insemination device that measures the reaction of cells suspended in a solution using a probe made of diamond or silicon carbide containing nitrogen-vacancy pairs, The system has an environmental control mechanism that changes the state of the cells by applying external stimuli to them, The aforementioned environmental control mechanism is The cells, suspended in the solution, are fixed while remaining suspended in the solution, and the external stimulus is applied to the cells. The aforementioned environmental control mechanism is A sample suction tube that determines the relative positional relationship between the cells suspended in the solution and the probe, and fixes the cells in the solution while keeping them suspended so as not to move, It has an injection tube that is positioned to be inserted into the cell and to inject a substance into the cell, The sample suction tube and the injection tube are, An artificial insemination device characterized by being arranged so as to face the nitrogen-vacancy pair at a predetermined angle.

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