Intracellular quantum metrology device, intracellular quantum metrology method, and cell retention device

Abstract

The purpose of the present invention is to achieve quantum metrology which makes it possible to measure the state of the inside of a cell and in which disadvantages of quantum metrology using nano-particles are overcome. The intracellular quantum metrology device (1) is provided with a substrate (10) having a nanopillar (11) formed on the surface thereof, in which the nanopillar (11) is intended to be inserted into a cell (C) and includes a quantum sensor at the tip part thereof.

Description

Technical Field

[0001] The present invention relates to an intracellular quantum measurement device and an intracellular quantum measurement method for measuring the state inside a cell using a quantum sensor. The present invention also relates to a cell holding device including such an intracellular quantum measurement device.

Background Art

[0002] When a nitrogen atom exists in a diamond crystal, a vacancy may be formed next to it. At this time, the NV (Nitrogen-Vacancy) center, which is the center of the nitrogen atom and the vacancy, has the property that its quantum state changes according to the surrounding environment such as an electric field, a magnetic field, and temperature, and the property that its quantum state can be read by fluorescence measurement. Therefore, the NV center can be used as a quantum sensor for detecting the surrounding environment. As a fluorescence microscope for performing fluorescence measurement, for example, those described in Patent Documents 1 and 2 are known.

[0003] Such a quantum sensor can be used to measure the state of a cell. For example, diamond nanoparticles containing an NV center can measure the state inside a cell if they are introduced into the cell, and are expected to be used as a "next-generation biomarker". In addition, a diamond nanofilm containing an NV center can measure the state of the cell membrane if it is attached to the cell membrane, and is expected to be used as a "next-generation coverslip".

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, while quantum measurement using nanoparticles (next-generation biomarkers) has the advantage of being able to measure the intracellular state, it has drawbacks such as (1) difficulty in precisely setting the measurement environment, (2) weak luminescence intensity, (3) poor spin characteristics, and (4) non-uniform characteristics for each quantum sensor (NV center). The main cause of these drawbacks is that the position and orientation of the quantum sensor cannot be fixed. As a result, for example, it becomes difficult to precisely adjust the direction of the magnetic field applied to the quantum sensor or to optimally select the pulse shape of the excitation light irradiated to the quantum sensor. Therefore, as a method of quantum measurement, DC measurement (measurement that captures changes in luminescence intensity using the luminescence state) is used.

[0006] On the other hand, quantum measurement using a diamond thin film (next-generation coverslip) has advantages such as (1) ease of precisely setting the measurement environment, (2) strong luminescence intensity, (3) good spin characteristics, and (4) uniform characteristics for each quantum sensor (NV center), but has the drawback of being unable to measure the intracellular state. The main cause of these advantages is that the position and orientation of the quantum sensor can be fixed. As a result, for example, it becomes easier to precisely adjust the direction of the magnetic field applied to the quantum sensor or to optimally select the pulse shape of the excitation light irradiated to the quantum sensor. Therefore, as a method of quantum measurement, AC measurement (measurement that captures the change in the phase of the quantum level using the superposition state) is used.

[0007] One aspect of the present invention has been made in view of the above problems, and its object is to realize quantum measurement capable of measuring the intracellular state and overcoming the drawbacks of quantum measurement using nanoparticles.

Means for Solving the Problems

[0008] In order to solve the above problems, one aspect of the present invention provides an intracellular quantum measurement device, characterized in that it comprises a nanopillar for insertion into a cell, and a substrate on the surface of which is formed a nanopillar including a quantum sensor at the tip.

[0009] In addition, in order to solve the above-mentioned problems, an intracellular quantum measurement method according to one aspect of the present invention is characterized in that it uses a substrate on the surface of which nanopillars containing quantum sensors at their tips are formed, and quantum measurement is performed with the nanopillars inserted into a cell. Effect of the Invention

[0010] According to one aspect of the present invention, quantum measurement capable of measuring intracellular states can be realized, which overcomes the disadvantages of quantum measurement using nanoparticles. [Brief description of the drawings]

[0011]

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Mode for Carrying Out the Invention

[0012] 〔Intracellular Quantum Measurement Device〕 The intracellular quantum measurement device 1 according to an embodiment of the present invention will be described with reference to FIG. 1. In FIG. 1, (a) is a plan view of the intracellular quantum measurement device 1, and (b) is a cross-sectional view of the intracellular quantum measurement device 1. In FIG. 1(b), the AA' cross-section (see FIG. 1(a)) of the intracellular quantum measurement device 1 is shown.

[0013] The intracellular quantum measurement device 1 includes a substrate 10 on the surface of which a plurality of nanopillars 11 are formed. The nanopillars 11 are columnar or conical structures Constructed body (In this embodiment, a structure in which the base portion is conical and the tip portion is columnar) for insertion into the cell C. At the tip portion 11a of each nanopillar 11, a quantum sensor is included. The nanopillars 11 are periodically (at the lattice points of a square lattice in this embodiment) arranged on the surface of the substrate 10.

[0014] In this embodiment, the substrate 10 and the nanopillar 11 are made of diamond crystal. In addition, in this embodiment, the tip 11a of the nanopillar 11 contains an NV center consisting of one nitrogen atom and one vacancy adjacent to this nitrogen atom, and this NV center is used as a quantum sensor. In order to precisely adjust the magnetic field applied to the quantum sensor, the

[0111] direction of the diamond crystal is aligned with the substrate 1. 0 The normal direction of the surface is aligned with that of the

[0015] The height H of the nanopillar 11 and the diameter D of the tip 11a are determined so that intracellular measurements can be realized (such as the ability to penetrate cell membranes and adhere cells, including suspended cells), taking into consideration the physical condition of sufficiently high fluorescence extraction efficiency and the biological condition of sufficiently low toxicity to cells.

[0016] From the above viewpoints, the height H of the nanopillars 11 may be 1 μm or more and 20 μm or less, preferably 1 μm or more and 5 μm or less, more preferably 2 μm or more and 4 μm or less, and even more preferably 2.5 μm or more and 3.5 μm or less. In this embodiment, the height H of the nanopillars 11 is set to 3.2 μm.

[0017] From the above viewpoint, the diameter D of the tip 11a of the nanopillar 11 may be 100 nm or more and 1000 nm or less, preferably 100 nm or more and 500 nm or less, more preferably 200 nm or more and 400 nm or less, and even more preferably 250 nm or more and 350 nm or less. In this embodiment, the diameter D of the tip 11a of the nanopillar 11 is 300 nm.

[0018] Furthermore, from the viewpoint of sufficiently increasing the efficiency of extracting fluorescence, the thickness T of the substrate 10 is preferably 50 μm or less. In this embodiment, the thickness T of the substrate 10 is set to 40 μm.

[0019] In addition, the interval (center-to-center distance) D between adjacent nanopillars 11 is determined to be about half the size of the cell to be measured so as to increase the probability that any of the nanopillars 11 is inserted into the cells cultured in the cell holding device 2 described later.

[0020] From the above viewpoints, the interval D between adjacent nanopillars 11 is preferably 10 μm or more and 60 μm or less. For example, if the cell to be measured is a somatic cell of a mammal (about 20 μm to 50 μm), the interval D between adjacent nanopillars 11 is more preferably 10 μm or more and 25 μm or less. Further, if the cell to be measured is a germ cell of a mammal (about 50 μm to 80 μm), the interval D between adjacent nanopillars 11 is more preferably 25 μm or more and 40 μm or less. Also, if the cell to be measured is a plant cell (about 80 μm to 120 μm), the interval D between adjacent nanopillars 11 is more preferably 40 μm or more and 60 μm or less.

[0021] According to the intracellular quantum measurement device 1 according to the present embodiment, quantum measurement can be performed in a state where the tip portion 11a of the nanopillar 11 is inserted into the cell C. Here, quantum measurement refers to specifying the quantum state of the quantum sensor by measuring the fluorescence emitted from the quantum sensor, and estimating the state around the quantum sensor from the specified quantum state. In the present embodiment, estimating the state around the quantum sensor means, that is, estimating the state inside the cell C.

[0022] It should be noted here that the nanopillar 11 is not floating in the cell in isolation like a nanoparticle, but is formed on the surface of the substrate like a nanothin film. Therefore, similar to the quantum measurement using a diamond thin film, AC measurement can be performed, and thus the state inside the cell C can be precisely measured using high-sensitivity spin analysis techniques such as NMR and ESR. That is, the state inside the cell C can be measured while overcoming the demerits of the quantum measurement using nanoparticles.

[0023] In the present embodiment, a configuration is adopted in which a plurality of nanopillars 11 are formed on the surface of the substrate 10. However, the present invention is not limited to this. That is, a configuration in which a single nanopillar 11 is formed on the surface of the substrate 10 may be adopted. However, by adopting a configuration in which a plurality of nanopillars 11 are formed on the surface of the substrate 10 as in the present embodiment, a further effect that quantum measurements of a plurality of cells (for example, a plurality of cells constituting a living tissue) can be simultaneously performed is obtained. Such simultaneous measurement is performed in a state where the tip of each of the plurality of nanopillars 11 is inserted into each of the plurality of cells.

[0024] 〔Cell holding device〕 The cell holding device 2 including the intracellular quantum measurement device 1 described above will be described with reference to FIG. 2. In FIG. 2, (a) is a plan view of the cell holding device 2, and (b) is a cross-sectional view of the cell holding device 2. In FIG. 2(b), a BB' cross-section of the cell holding device 2 (see FIG. 2(a)) is shown.

[0025] The cell holding device 2 includes a first substrate 21, a second substrate 22, and a third substrate 23. The first substrate 21 is a substrate that functions as a coverslip. The material of the first substrate 21 is arbitrary, but for example, it is an insulator (dielectric) that transmits the excitation light irradiated to the quantum sensor and the fluorescence emitted from the quantum sensor. The second substrate 22 is a substrate on which an opening 221 functioning as a chamber is formed, and is laminated on the first substrate 21. The material of the second substrate 22 is arbitrary, but for example, it is an insulator (dielectric) with low thermal conductivity. The third substrate 23 is a substrate that functions as a lid of the chamber, and is laminated on the second substrate 22 after injecting a liquid 26 containing cells (pure water in the present embodiment) into the opening 221. The material of the third substrate 23 is arbitrary, but for example, it is an insulator (dielectric) with low thermal conductivity. In the present embodiment, quartz glass is used as the material of the first substrate 21, the second substrate 22, and the third substrate 23.

[0026] The cell holding device 2 further includes an intracellular quantum measurement device 1 (substrate 10 on which nanopillars 11 are formed), a first electrode 24a, and a second electrode 24b. The intracellular quantum measurement device 1 is laminated on the first substrate 21 inside the opening 221. Here, the intracellular quantum measurement device 1 is laminated on the first substrate 21 such that the back surface of the substrate 10 on which the nanopillars 11 are not formed is in surface contact with the surface of the first substrate 21. Note that the intracellular quantum measurement device 1 may be fixed to a frame to facilitate handling. In this case, the intracellular quantum measurement device 1 is fixed to the first substrate 21 via this frame.

[0027] The first electrode 24a and the second electrode 24b are each a strip-shaped conductor and are laminated on the first substrate 21. The first electrode 24a is laminated on the first substrate 21 such that its outer edge is along the first side of the intracellular quantum measurement device 1, and the second electrode 24b is laminated on the first substrate 21 such that its outer edge is along the second side (the side opposite to the first side) of the intracellular quantum measurement device 1. The first electrode 24a and the second electrode 24b are short-circuited by a conducting wire 24c that straddles the intracellular quantum measurement device 1 diagonally. The conducting wire 24c functions as an antenna that radiates an electromagnetic wave to be applied to the quantum sensor when a high-frequency current is input to the first electrode 24a and the second electrode 24b.

[0028] Openings 222a and 232a are formed in the second substrate 22 and the third substrate 23, respectively, so as to be able to access the first electrode 24a from the upper surface side of the cell holding device 2. Similarly, openings 222b and 232b are formed in the second substrate 22 and the third substrate 23, respectively, so as to be able to access the second electrode 24b from the upper surface side of the cell holding device 2. Thereby, a high-frequency current source can be easily connected to the first electrode 24a and the second electrode 24b.

[0029] Note that the width W of the first electrode 24a and the second electrode 24b is preferably 0.1 mm or more and 500 mm or less. Thereby, it becomes possible to apply an electromagnetic field (electromagnetic wave) in a wide frequency band from several MHz to several GHz to the cells in the liquid 26. In the present embodiment, assuming that an electromagnetic field (electromagnetic wave) from 1 MHz to 120 GHz is applied, the width W of the first electrode 24a and the second electrode 24b is set to 2.6 mm.

[0030] Also, the total T + U of the thickness U of the first substrate 21 of the cell holding device 2 and the thickness T of the substrate 10 of the intracellular quantum measurement device 1 is preferably 0.3 mm or less. Thereby, it becomes possible to irradiate excitation light and detect fluorescence using an objective lens widely used in fluorescence observation of cells. In the present embodiment, assuming that an objective lens with a working distance Wd of 0.3 mm is used, the thickness U of the first substrate 21 of the cell holding device 2 and the thickness T of the substrate 10 of the intracellular quantum measurement device 1 are set so that the total T + U is 0.21 mm.

[0031] By using the cell holding device 2 according to the present embodiment, the nanopillar 11 of the intracellular quantum measurement device 1 can be inserted into the cells cultured in the liquid 26, and the internal state of the cells can be easily measured. Moreover, by inputting a high-frequency current from the high-frequency current source to the first electrode 24a and the second electrode 24b, the internal state of the cells in the electromagnetic field (electromagnetic wave) can be easily measured.

[0032] Note that the first electrode 24a and the second electrode 24b do not have to be short-circuited. In this case, when a voltage is applied between the first electrode 24a and the second electrode 24b, an electric field corresponding to the voltage can be applied to the quantum sensor. Two electrodes 24b, an electric field corresponding to the voltage can be applied to the quantum sensor.

[0033] 〔Intracellular Quantum Measurement System〕 The intracellular quantum measurement system 3 including the above-described cell holding device 2 will be described with reference to FIG. 3. FIG. 3 is a configuration diagram of the intracellular quantum measurement system 3.

[0034] The intracellular quantum measurement system 3 includes a cell holding device 2, a housing 30, a light emitter 31, a light receiver 32, a single photon counter 33, first to sixth lenses L1 to L6, first to third mirrors M1 to M3, and a pinhole P. The intracellular quantum measurement system 3 has a wide-field microscope mode and a confocal microscope mode. To switch between these modes, the first lens L1 and the third mirror M3 are movable.

[0035] The housing 30 is configured to house the first to third lenses L1 to L3 and the first to third mirrors M1 to M3. In this embodiment, the housing 30 is the housing of an existing inverted microscope.

[0036] The light emitting unit 31 is configured to emit excitation light to be irradiated onto the quantum sensor. In this embodiment, the light emitting unit 31 is an LED that emits laser light with a wavelength of 532 nm.

[0037] On the other hand, in the wide-field microscope mode, the first lens L1 is inserted into the optical path of the excitation light, and the third mirror M3 is removed from the optical path of the fluorescence. Therefore, the excitation light (collimated light) from the light emitter 31 is converted into converging light by the first lens L1, then reflected by the first mirror M1, and enters the second lens L2. The second lens L2 converts the converging excitation light into collimated light, and irradiates it planarly onto the intracellular quantum measurement device 1 (more precisely, the surface of the substrate 10) in the cell holding device 2. The fluorescence (diverging light) from the intracellular quantum measurement device 1 is collimated by the second lens L2, then reflected by the second mirror M2. The fluorescence reflected by the second mirror M2 is collected by the third lens L3 and enters the light receiving unit 32.

[0038] In the confocal microscope mode, the first lens L1 is removed from the optical path of the excitation light, and the third mirror M3 is placed on the optical path of the fluorescence. Therefore, the excitation light (collimated light) from the light emitting unit 31 is reflected by the first mirror M1 and enters the second lens L2. The second lens L2 converts the excitation light, which is collimated light, into convergent light and irradiates it pointwise onto the intracellular quantum measurement device 1 (more precisely, the surface of the substrate 10) in the cell holding device 2. The fluorescence (divergent light) from the intracellular quantum measurement device 1 is collimated by the second lens L2 and then reflected by the third mirror M3. The fluorescence reflected by the third mirror M3 is converted into convergent light by the fourth lens L4, passes through the pinhole P, and is then collimated by the fifth lens L5. Then, it is focused by the sixth lens L6 and enters the single photon counter 33.

[0039] Note that the first mirror M1 needs to reflect the excitation light and transmit the fluorescence. Therefore, in this embodiment, a dichroic mirror is used as the first mirror M1. The first mirror M1 receives the fluorescence from the intracellular quantum measurement device 1 and also the excitation light reflected by the cell holding device 2. However, since this excitation light is reflected by the first mirror M1, it is not guided to the light receiving unit 32.

[0040] The light receiving unit 32 is configured to receive the fluorescence emitted from the quantum sensor in the wide-field microscope mode. In this embodiment, a high-sensitivity imaging camera, specifically, an EMCCD (Electron Multiplying Charge Coupled Device), is used as the light receiving unit 32. Single photon Counter 33 is configured to receive the fluorescence emitted from the quantum sensor in the confocal microscope mode. In this embodiment, single photon Counter 33 as 、A a PD (Avalanche Photodiode) is used.

[0041] When measuring the intracellular state using the intracellular quantum measurement system 3, in each measurement cycle, the light emitting unit 31 outputs one pulse of excitation light, and the light receiving unit 32 detects one pulse of fluorescence output from the quantum sensor as its response. The time required for each measurement cycle is about 5 μs. In contrast, the frame rate of the video output from the high-sensitivity image sensor is about 50 frames / second. That is, the Sensitivity time required to read out one image from the image sensor (hereinafter also referred to as the "readout cycle") is about 20 ms, which is about 4000 times the time required for each measurement cycle. Therefore, in the intracellular quantum measurement system 3, N measurement cycles (N is a natural number of 2 or more, and in this embodiment, N is about 1000) are executed in each readout cycle, and a configuration is adopted to multiply the effective sensitivity of the high-sensitivity image sensor by N.

[0042] According to the intracellular quantum measurement system 3 according to this embodiment, it is possible to achieve both wide-field measurement in the wide-field microscope mode and high-resolution and high-sensitivity measurement in the confocal microscope mode. Moreover, according to the intracellular quantum measurement system 3 according to this embodiment, the wide-field microscope mode and the confocal microscope mode can be easily switched without touching the measurement target.

[0043] Note that the intracellular quantum measurement system 3 may further include a magnetic field fixing device 34. In quantum measurement, it is necessary to adjust the direction of the magnetic field acting on the quantum sensor with an accuracy of 10 degrees or less. By using the magnetic field fixing device 34, the direction of the magnetic field acting on the quantum sensor can be accurately adjusted in four directions (three-axis directions + rotation direction). Such adjustment is realized, for example, by performing ODMR (Optically Detected Magnetic Resonance) measurement while changing the direction of the magnetic field using the magnetic field fixing device 34 and determining the direction of the magnetic field with reference to the contrast and fluorescence intensity of the measurement results. Thereby, for example, it becomes possible to accurately apply a high magnetic field, which is important for achieving high frequency resolution in structural analysis such as NMR and ESR.

[0044] [Supplement on Effects] According to the intracellular quantum measurement system 3, optimized fluorescence extraction can be performed in a state where a quantum measurement material is introduced into a biological measurement sample in various forms. Further, by using a general-purpose computer (not shown) in combination, ODMR (Optically Detected Magnetic Resonance) measurement can be performed after precisely controlling environments such as the direction of a magnetic field, temperature, and humidity. Here, ODMR measurement is a measurement method for measuring a change in the quantum state of a quantum sensor by performing irradiation with excitation light and measurement of fluorescence intensity while changing the frequency of microwaves. In this case, the intracellular quantum measurement device 1 is used to introduce a quantum sensor into a cell, the cell holding device 2 is used to apply microwaves to the quantum sensor introduced into the cell, and the intracellular quantum measurement system 3 is used to detect the intensity of fluorescence emitted from the quantum sensor introduced into the cell. Further, in ODMR measurement, a computer (not shown) is used to control the timing of irradiating excitation light and the timing of applying microwaves, and to calculate the quantum state of the quantum sensor from the detected fluorescence intensity.

[0045] In addition, by performing application of microwaves and detection of fluorescence intensity with a time resolution of 400 ps or less, advanced quantum operations such as rabi and echo can be realized. Further, by increasing the number of microwaves applied in microregion spin measurement and controlling spins other than the quantum sensor, DEER (Double Electron Electron Resonance) can be realized.

[0046] [Examples] The inventors performed intracellular quantum measurement of Hepa1-6 cells using the intracellular quantum measurement system 3. As the intracellular quantum measurement device 1, one in which nanopillars 11 having a height of 3.2 μm and a diameter of 300 nm were periodically formed at intervals of 15 μm on the surface of a substrate having a thickness of 40 μm was used.

[0047] (a) of FIG. 4 is an image obtained by imaging the surface of the intracellular quantum measurement device 1. (b) of FIG. 4 is an image obtained by imaging the nanopillars 11 of the intracellular quantum measurement device 1. (c) and (d) of FIG. 4 are images obtained by imaging Hepa1-6 cells cultured in the cell holding device 2. The Hepa1-6 cells were pre-stained using a cell membrane staining reagent. As the cell membrane staining reagent, CellMask (registered trademark) Green was used. From (c) and (d) of FIG. 4, it can be seen that the nanopillars 11 are inserted into the Hepa1-6 cells.

[0048] The inventors performed Rabi measurement in a state where the nanopillars 11 were inserted into cells using the intracellular quantum measurement system 3. FIG. 5 is a graph showing the τ-dependency of the normalized contrast obtained by this Rabi measurement. Here, the normalized contrast is a dimensionless quantity representing the state transition of electron spins (|0> and |±1> using the NV center as a quantum bit) by microwave operation. Also, τ is a quantity representing the microwave application time, and the unit is nanoseconds. From the graph shown in FIG. 5, it can be seen that the state transition of electron spins corresponding to the microwave application time is realized. Thereby, it was confirmed that quantum operation with high contrast can be performed inside cells.

[0049] The inventors also performed NMR measurement in a state where the nanopillars 11 were inserted into cells using the intracellular quantum measurement system 3. FIG. 6 is a graph showing the τ-dependency of the normalized contrast obtained by this NMR measurement. From the graph shown in FIG. 6, it can be seen that peaks (dips) corresponding to nuclear spins present inside the cell appear in the region where τ is 164 nanoseconds or more and 171 nanoseconds or less. Even when NMR measurement is performed in a state where the nanopillars 11 are not inserted into the cells, such peaks (dips) do not appear. Thereby, it was confirmed that quantum measurement of the state inside cells can be realized using the nanopillars 11.

[0050] In addition, the inventors measured the time-dependence of the normalized contrast with the intracellular quantum measurement system 3 while the nanopillar 11 was inserted into the cell. FIG. 7 is a graph showing the time-dependence of the normalized contrast obtained by this measurement. From the graph shown in FIG. 7, it can be seen that the coherence time (T2) is 15.9 μs. Thus, it was confirmed that the quantum property of the quantum sensor contained at the tip of the nanopillar 11 inserted into the cell is maintained for a sufficiently long time.

[0051] As described above, the inventors enabled the detection of nuclear spins in minute regions within cells by using the nanopillar 11. In addition to the quantum operation of the quantum sensor, the inventors also performed a quantum operation on the electron spins near the quantum sensor. This will be described in detail below.

[0052] The inventors performed an operation (two-quantum operation) on the quantum state of the quantum sensor (NV center) inserted into the cell and the electron spins (quantum states) of molecules, atoms, ions, or radicals (hereinafter referred to as "radicals, etc.") within the cell that exist around the tip portion 11a of the nanopillar 11 inserted into the cell.

[0053] FIG. 8(a) is a diagram showing the nanopillar 11, and FIG. 8(b) is a schematic diagram showing an enlarged view of the quantum sensor (NV center) disposed at the tip portion 11a of the nanopillar 11. In this embodiment, the height H of the nanopillar 11 is 3.2 μm, and the diameter D of the tip portion 11a of the nanopillar 11 is 300 nm. The NV center is disposed at a depth of about 10 nm from the surface of the tip portion 11a of the nanopillar 11 and can detect the quantum operation of surface radicals near the tip portion 11a (quantum sensor). Here, the periphery described above includes the periphery of each of the bulged Curved surface portion that constitutes the foremost end surface of the tip portion 11a and the outer peripheral surface that constitutes the cylindrical side surface of the tip portion 11a.

[0054] FIG. 9 is a diagram showing a sequence for quantum operation of a quantum sensor and radicals or the like in the vicinity of the quantum sensor in the nanopillar 11 of FIG. 8. The sequence SQ1 represents a pulse sequence for applying microwaves to the quantum sensor (NV center), and the sequence SQ2 represents a pulse sequence for applying microwaves to radicals or the like (electron spins). The sequence SQ3 represents a pulse sequence for laser readout.

[0055] These sequences SQ1 to SQ3 can be considered to correspond to the following processes (1) to (3), respectively. That is, by the processes (1) to (3), an intracellular quantum measurement method can be configured. (1) A first quantum operation process of applying a pulse of a first electromagnetic wave (for example, microwaves) to a quantum sensor (NV center) to operate the quantum state of the quantum sensor (2) A second quantum operation process of applying a pulse of a second electromagnetic wave (for example, microwaves) having a frequency different from that of the first electromagnetic wave to radicals or the like (at least any one of molecules, atoms, ions, radicals) existing around the tip 11a to operate the quantum state (electronic state) of the radicals or the like (3) A state detection process of irradiating the quantum sensor and radicals or the like with excitation light (for example, a pulse of laser light) and receiving fluorescence from the quantum sensor and radicals or the like

[0056] In the sequence SQ1, a pulse of microwaves is applied to the NV center at a time interval τ to switch the state (π / 2, π) of the NV center. In the sequence SQ2, a pulse of microwaves with a spin operation time (pulse width) Tau is applied to the external electron spin of radicals or the like. These microwaves (electromagnetic waves) are applied from the conducting wire 24c (antenna) as described above.

[0057] In the sequence SQ3, for example, laser light (excitation light) with a pulse width of 300 to 500 ns is irradiated, and fluorescence from the quantum sensor and radicals or the like is received. As described above, the excitation light is irradiated from the light emitting unit 31, and the fluorescence is received by the single photon counter 33 or the light receiving unit 32.

[0058] Sequences SQ1 to SQ3 can be controlled independently of each other. By controlling the timing of sequences SQ1 to SQ3, operations on electron spins such as radicals near the quantum sensor can be confirmed.

[0059] Figure 10 is a graph showing an example of the results obtained using the pulse sequence of Figure 9, and represents the Tau dependence of the contrast obtained by ESR measurement using two-qubit operations. In graphs Fit1 and Fit2, the intensities of the microwaves applied to the radicals are different from each other. The intensity of the microwave applied to the radical is greater in graph Fit1 than in graph Fit2. In both graphs Fit1 and Fit2, the rotational motion of the radical is measured by changing the spin operation time Tau. By decreasing the intensity of the microwave applied from graph Fit1 to graph Fit2, it can be seen that the oscillation period called Rabi oscillation decreases. That is, it can be confirmed that an ESR reaction is detected for the microwave and quantum operations are being performed.

[0060] Measurement and quantum operations of electron spins in a minute region (for example, a volume of about (5 nm) 3 have hitherto been used for the detection of single proteins formed on a thin film and the analysis of their structures, and for the detection of single DNA and the analysis of its structure. However, such measurements and quantum operations have not been performed inside cells. The inventors have made it possible to detect surface radicals and perform quantum operations by forming a single NV center at the tip of a diamond pillar.

[0061] The inventors' quantum measurement method for quantum-operating both a quantum sensor and radicals, etc., enables various measurements inside cells. Examples thereof include the measurement of single DNA, the structural analysis of Cryptochrome protein related to the detection of the direction of migratory birds, the identification and detection of ions, and the structural analysis of various radicals generated in intracellular oxidation phenomena.

[0062] 〔Summary〕 The intracellular quantum measurement device according to Aspect 1 includes a substrate on which a nanopillar for insertion into a cell is formed on the surface, and the nanopillar includes a quantum sensor at its tip.

[0063] According to the above configuration, it is possible to perform quantum measurement capable of measuring the state inside a cell, and realize quantum measurement that overcomes the demerits of quantum measurement using nanoparticles.

[0064] In the intracellular quantum measurement device according to Aspect 2, in addition to the configuration of the intracellular quantum measurement device according to Aspect 1, a configuration is adopted in which the height of the nanopillar is 1 μm or more and 20 μm or less.

[0065] According to the above configuration, it is possible to realize intracellular quantum measurement while satisfying both the physical condition of making the fluorescence extraction efficiency sufficiently high and the biological condition of making the toxicity to cells sufficiently low.

[0066] In the intracellular quantum measurement device according to Aspect 3, in addition to the configuration of the intracellular quantum measurement device according to Aspect 1 or 2, a configuration is adopted in which the diameter of the tip of the nanopillar is 100 nm or more and 1000 μm or less.

[0067] According to the above configuration, it is possible to realize intracellular quantum measurement while satisfying both the physical condition of making the fluorescence extraction efficiency sufficiently high and the biological condition of making the toxicity to cells sufficiently low.

[0068] In the intracellular quantum measurement device according to Aspect 4, in addition to the configuration of the intracellular quantum measurement device according to any one of Aspects 1 to 3, the substrate has a plurality of nanopillars for insertion into each of a plurality of cells, and a plurality of nanopillars including quantum sensors at their tips are formed on the surface.

[0069] According to the above configuration, it is possible to simultaneously measure the states inside a plurality of cells.

[0070] In the intracellular quantum measurement device according to Aspect 5, in addition to the configuration of the intracellular quantum measurement device according to Aspect 4, the distance between the adjacent nanopillars is 10 μm or more and 60 μm or less, and such a configuration is adopted.

[0071] According to the above configuration, the nanopillars can be inserted into cells more reliably. As a result, the state inside the cells can be quantum-measured more reliably.

[0072] In the intracellular quantum measurement device according to Aspect 6, in addition to the configuration of the quantum measurement device according to any one of Aspects 1 to 5, the nanopillars and the substrate are made of diamond crystal, and the quantum sensor is an NV center, and such a configuration is adopted.

[0073] According to the above configuration, the state inside the cells can be quantum-measured accurately.

[0074] In the intracellular quantum measurement device according to Aspect 7, in addition to the configuration of the quantum measurement device according to Aspect 6, the

[0111] direction of the diamond crystal coincides with the normal direction of the surface of the substrate, and such a configuration is adopted.

[0075] According to the above configuration, the magnetic field applied to the quantum sensor inside the cells can be controlled accurately.

[0076] The cell holding device according to Aspect 8 includes a first substrate, a second substrate laminated on the first substrate, the second substrate having an opening formed as a chamber for storing a liquid containing cells, and an intracellular quantum measurement device according to any one of Aspects 1 to 7 laminated on the first substrate in the opening, and an electrode laminated on the intracellular quantum measurement device in the opening.

[0077] According to the above configuration, the state inside the cells cultured in the liquid stored in the chamber can be easily quantum-measured.

[0078] The intracellular quantum measurement method according to Aspect 9 uses a substrate on which a nanopillar containing a quantum sensor at its tip is formed on the surface, and performs quantum measurement with the nanopillar inserted into a cell.

[0079] According to the above configuration, it is possible to perform quantum measurement capable of measuring the state inside a cell, and it is possible to realize quantum measurement that overcomes the demerits of quantum measurement using nanoparticles.

[0080] The intracellular quantum measurement method according to Aspect 10, in addition to the configuration of the intracellular quantum measurement method according to Aspect 9, uses a substrate on which a plurality of nanopillars each containing a quantum sensor at its tip are formed on the surface, and performs quantum measurement with each of the plurality of nanopillars inserted into each of the plurality of cells.

[0081] According to the above configuration, it is possible to simultaneously measure the states inside a plurality of cells.

[0082] The intracellular quantum measurement method according to Aspect 11, in addition to the configuration of the intracellular quantum measurement method according to Aspect 9 or 10, includes a first quantum operation process of applying a pulse of a first electromagnetic wave to the quantum sensor to operate the quantum state of the quantum sensor, a second quantum operation process of applying a pulse of a second electromagnetic wave having a frequency different from that of the first electromagnetic wave to at least any one of molecules, atoms, ions, and radicals existing around the tip portion to operate the quantum state of at least any one of the molecules, the atoms, the ions, and the radicals, and a state detection process of irradiating excitation light to the quantum sensor and at least any one of the molecules, the atoms, the ions, and the radicals, and receiving fluorescence from the quantum sensor and at least any one of the molecules, the atoms, the ions, and the radicals.

[0083] According to the above configuration, by performing quantum operations on both the quantum sensor and radicals, etc., various information can be obtained.

[0084] (Supplementary Notes) The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

Description of Reference Numerals

[0085] 1 Intracellular quantum measurement device 10 Substrate 11 Nanopillar 2 Cell holding device 3 Intracellular quantum measurement system

Claims

1. A nanopillar for insertion into a cell, comprising a substrate having a nanopillar with a quantum sensor formed on its tip on the surface. An intracellular quantum measurement device characterized by this.

2. The height of the nanopillar is 1 μm or more and 20 μm or less. The intracellular quantum measurement device according to Claim 1, characterized by this.

3. The diameter of the tip of the nanopillar is 100 nm or more and 1000 μm or less. The intracellular quantum measurement device according to Claim 1 or 2, characterized by this.

4. On the substrate, there are a plurality of nanopillars for insertion into each of a plurality of cells, and a plurality of nanopillars with a quantum sensor formed on their tips are formed on the surface. The intracellular quantum measurement device according to Claim 1 or 2, characterized by this.

5. The distance between adjacent nanopillars is 10 μm or more and 60 μm or less. The intracellular quantum measurement device according to Claim 4, characterized by this.

6. The nanopillar and the substrate are composed of a diamond crystal, and the quantum sensor is an NV center. The intracellular quantum measurement device according to Claim 1 or 2, characterized by this.

7. The [111] direction of the diamond crystal coincides with the normal direction of the surface of the substrate. The intracellular quantum measurement device according to Claim 6, characterized by this.

8. A first substrate, a second substrate laminated on the first substrate, the second substrate having an opening formed as a chamber for storing a liquid containing cells, the intracellular quantum measurement device according to Claim 1 or 2 laminated on the first substrate within the opening, and an electrode laminated on the intracellular quantum measurement device within the opening. A cell holding device characterized by this.

9. Using a substrate having a nanopillar with a quantum sensor formed on its tip, performing quantum measurement with the nanopillar inserted into a cell. An intracellular quantum measurement method characterized by this.

10. Using a substrate having a plurality of nanopillars with a quantum sensor formed on their tips, performing quantum measurement with the plurality of nanopillars inserted into each of a plurality of cells respectively. The intracellular quantum measurement method according to Claim 9, characterized by this.

11. Applying a pulse of a first electromagnetic wave to the quantum sensor to perform a first quantum operation process for operating the quantum state of the quantum sensor. Applying a pulse of a second electromagnetic wave, which has a frequency different from that of the first electromagnetic wave, to at least any one of molecules, atoms, ions, and radicals existing around the tip portion to perform a second quantum operation process of manipulating the quantum state of at least any one of the molecules, the atoms, the ions, and the radicals. A state detection process of irradiating the quantum sensor and at least any one of the molecules, the atoms, the ions, and the radicals with excitation light and receiving fluorescence from the quantum sensor and at least any one of the molecules, the atoms, the ions, and the radicals. The intracellular quantum measurement method according to claim 9 or 10, characterized by the above.