ODMR temperature measurement method

Abstract

This invention addresses the problem of providing a technique that makes it possible to more accurately measure temperature on the basis of optically detected magnetic resonance. To address this problem, provided is a method for measuring the temperature of an object on the basis of the optically detected magnetic resonance of inorganic fluorescent particles, comprising: (a) a step for emitting microwaves of a plurality of different frequencies onto an object including inorganic fluorescent particles, (b) a step for using separate photon counters to respectively measure the fluorescence intensities of the inorganic fluorescent particles when each of the microwaves are emitted, (c) a step for correcting the fluorescence intensities on the basis of pulse measurement number discrepancy between the photon counters, and (d) a step for using the obtained corrected values to calculate the temperature of the object.

Description

[Technical field]

[0001] The present invention relates to an ODMR (Optically Detected Magnetic Resonance) temperature measurement method and the like. [Background technology]

[0002] Various technologies for measuring the temperature inside cells have been developed based on fluorescence detection. Examples of such technologies include a technology that uses fluorescent dyes or fluorescent polymer nanoparticles to measure temperature based on the change in wavelength peak of the fluorescence spectrum, a technology that uses quantum dots to measure temperature based on the change in wavelength peak of the fluorescence spectrum, and a technology that uses inorganic fluorescent particles such as fluorescent nanodiamonds to measure temperature based on the change in frequency peak of the photodetected electron spin resonance spectrum. Although these technologies have relatively high spatial resolution and temperature sensitivity, they have not been able to measure temperature inside living organisms.

[0003] On the other hand, in vivo temperature measurement techniques using fluorescent dyes and rare earth nanoparticles have been reported in mice, but these have low spatial resolution and temperature sensitivity, making it impossible to measure temperature at the single-cell level. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2014 / 165505 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventor has focused on a temperature measurement method based on the optical detection electron spin resonance spectrum peak shift and has been conducting research on this. In the past, a method was reported in which the entire spectrum peak was captured and the peak shift was calculated. However, this method required time to capture the entire spectrum peak, making real-time temperature measurement difficult. To solve this problem, Patent Document 1 proposes a technique in which, instead of capturing the entire estimated spectrum peak, four points in the peak are measured and the peak shift is calculated from the obtained measurement values.

[0006] In further research into this multi-point measurement technology, the inventors discovered that when performing real-time temperature measurement in a dynamic environment such as at the cellular or individual level, fluctuations in the number of measured photons occur, which causes artifacts in the temperature measurement. Due to such artifacts, the measured temperature fluctuates significantly even when the actual temperature is constant.

[0007] Therefore, an object of the present invention is to provide a technique that can measure temperature with higher accuracy based on optically detected magnetic resonance. Preferably, an object of the present invention is to provide a technique that can measure temperature changes in a cell or an individual in real time with higher accuracy. [Means for solving the problem]

[0008] As a result of intensive research, the inventor has found that there is a difference in photocount response (error in the number of pulses derived from photons) between multiple photon counters used in multipoint measurement, which causes the above-mentioned artifacts in the temperature measurement value. Based on this finding, the inventor has further researched and found that the following method, that is, a method for measuring the temperature of an object based on the optically detected magnetic resonance of inorganic fluorescent particles, includes the steps of (a) irradiating an object containing inorganic fluorescent particles with multiple types of microwaves having different frequencies from each other, (b) measuring the fluorescence intensity of the inorganic fluorescent particles when irradiated with each type of microwave using separate photon counters, (c) correcting the fluorescence intensity based on the error in the number of pulses measured between the photon counters, and (d) calculating the temperature of the object based on the obtained correction value, can measure the temperature with higher accuracy. Based on this finding, the inventor has further researched and completed the present invention.

[0009] That is, the present invention includes the following aspects.

[0010] Item 1. A method for measuring the temperature of an object based on optically detected magnetic resonance of inorganic fluorescent particles, comprising: (a) irradiating a target object containing inorganic fluorescent particles with a plurality of microwaves having different frequencies; (b) measuring the fluorescence intensity of the inorganic fluorescent particles when irradiated with various microwaves using separate photon counters; (c) correcting the fluorescence intensity based on the error in the number of pulses counted between the photon counters; and (d) calculating the temperature of the object based on the obtained correction value; Methods including. Item 2. The method according to Item 1, wherein the inorganic fluorescent particles are NV center-containing diamond. Item 3. The method according to Item 1 or 2, wherein the plurality of types of microwaves are 2 to 10 types. Item 4. The method according to any one of Items 1 to 3, wherein the multiple types of microwaves are six types. Item 5. The method according to any one of Items 1 to 4, wherein the inorganic fluorescent particles are tracked during measurement. Item 6. The method according to any one of Items 1 to 5, wherein the object is a cell, a microorganism, or an organoid. Item 7. The method according to any one of Items 1 to 6, wherein a change in temperature of the object over time is measured. Item 8. The method according to any one of items 1 to 7, further comprising measuring a temperature change when a stimulus is applied to the object. Item 9. The method according to any one of Items 1 to 8, wherein the step (c) includes a step of subtracting a previously measured value of an error in the number of pulses counted between the photon counters from the measured value of either one of the two corresponding fluorescence intensities, or adding the value to the measured value of either one of the two corresponding fluorescence intensities.

[0011] Item 10. The plurality of types of microwaves are six types, and The step (d) calculates the correction value obtained in the step (c) according to the following formula:

[0012]

number

[0013] [In the formula, α indicates the temperature dependence of the luminescence center (NV). δω indicates the frequency difference between the first and third, or the fourth and sixth, frequencies from the low frequency side of the microwave. I1 to I6 indicate the correction values ​​obtained by irradiating six types of microwaves, respectively.] Substituting the temperature change at the luminescence center (δT NV Item 10. The method according to any one of items 1 to 9, comprising a step of calculating

[0014] Item 11. A temperature measuring device that measures the temperature of an object based on optically detected magnetic resonance of inorganic fluorescent particles, comprising (A) a microwave irradiation device, (B) a photon counter, (C) a calculation unit that corrects the fluorescence intensity, and (D) a calculation unit that calculates the temperature. Item 12. The temperature measuring device of claim 11, further comprising: (E) a particle tracking system. Effect of the Invention

[0015] According to the present invention, it is possible to provide a technique that can measure temperature with higher accuracy based on optically detected magnetic resonance. [Brief description of the drawings]

[0016] [Figure 1] 1A is a graph showing an example of an ODMR spectrum, and FIG. 1B is a graph showing an example of the temperature dependence of the peak value of the ODMR spectrum. [Diagram 2] FIG. 1 is a schematic diagram of an ODMR spectrum peak. [Diagram 3] A schematic diagram of the optical arrangement and microwave circuitry in the apparatus used for ODMR temperature measurements in the examples is shown. NDF: Neutral density filter. LLF: Laser line filter. HWP: Half wave plate. L: Lens. DBS: Dichroic beam splitter. LPF: Long pass filter. CCD: Charge coupled device camera. BS: Beam splitter. APD: Avalanche photodiode. SPA: Spectrum analyzer. MW: Microwave source. DAQ: Data acquisition board. SpinCore: Bit pattern generator. [Figure 4] The variation of the photoresponse of the counters is shown. (a) The photon counts of each counter, I1 through I6, as a function of the laser excitation power of the ND fluorescence. (b) The difference in photon counts between two sets of counters, i.e. (I1, I6), (I2, I5), and (I3, I4), as a function of I1, I2, and I3, respectively. The solid lines are second order polynomial fits to the data. [Diagram 5]Development and characterization results of a real-time fast temperature measurement method are shown. (a) Time profile of photon counts for all counters (top) over 200 s with fluorescence intensity artifacts varied at a sampling rate of 500 ms. The corresponding estimated temperature profile of the ND-NV center is shown both without (middle) and with (bottom) counter calibration. Grey: TNV every 1 s. Red: adjacent average of 34 s. (b) Time profiles of ambient air temperature (TAir, top), total photon count detected (middle), and TNV (bottom) for a step change in temperature of the microscope objective heater (TOBJ). Inset is a close-up of the graph of a transient temperature rise of 0.7 °C with a time constant of 19 s. Grey: TNV every 1 s, red: adjacent average of 34 s, blue: TOBJ. (c) Close-up of the RMS (bottom) of TNV (top) and (TNV-TOBJ) for the period from 38 to 140 min. The precision is 0.3°C and is shown by the solid line. [Figure 6] Micrographs of ND-labeled C. elegans worms are shown in (a) DIC, (b) green (c) red confocal fluorescence, and (d) merged images. [Figure 7] In vivo temperature measurements of a C. elegans during environmental temperature change. (a) Shows an integrated photograph of the worm's NDs near the internal larvae. The yellow arrow indicates the ND used for temperature measurement. The black shadow displayed at the bottom is a copper-wire microwave linear antenna. (b) Shows the CW-ODMR spectrum of the ND. (c) Shows the time profile of photon count and TNV during environmental change. The inset shows a close-up view of the photon counts with frequent relocation every 4 seconds. Approximately every minute, a large position correction is seen. Grey: TNV every second, red: 34 seconds adjacent average, blue: TOBJ. (d) Shows the integrated photograph after the measurement. (e) Shows the RMS of (TNV-TOBJ) against the temperature change. The precision is 0.6 °C and is indicated by the solid line. [Figure 8]The temperature rise of worms due to biochemical stimulation with FCCP is shown. (a) Photon counts (top) and TNV (bottom) during FCCP stimulation are shown. The dashed blue line is the baseline estimated from the control experiment. The inset shows the baseline-subtracted TNV. (b) Merged bright-field and red-fluorescence photographs taken at multiple time points indicated as 1-5 in (a) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0018] In one aspect, the present invention relates to a method for measuring the temperature of an object based on the optically detected magnetic resonance of inorganic fluorescent particles, the method including: (a) a step of irradiating an object containing inorganic fluorescent particles with multiple types of microwaves having different frequencies from each other; (b) a step of measuring the fluorescence intensity of the inorganic fluorescent particles when irradiated with each type of microwave using separate photon counters; (c) a step of correcting the fluorescence intensity based on the error in the number of pulses measured between the photon counters; and (d) a step of calculating the temperature of the object based on the obtained correction value (also referred to as the "measurement method of the present invention" in this specification). This will be described below.

[0019] 1. Temperature measurement based on optically detected magnetic resonance The measurement method of the present invention is a method for measuring the temperature of an object based on optically detected magnetic resonance (hereinafter, sometimes referred to as "ODMR") of inorganic fluorescent particles. ODMR is as follows.

[0020] Inorganic fluorescent particles absorb microwaves at their resonant frequency and exhibit electron spin resonance. During electron spin resonance, non-radiative energy deactivation increases in the excited electronic state. Therefore, when microwaves are irradiated, the amount of fluorescence decreases compared to when microwaves are not irradiated. For the NV center in diamond, when the external magnetic field is zero, electron spin resonance occurs at a frequency F of 2.87 GHz.

[0021] FIG. 1(a) is a graph showing an example of an ODMR spectrum. FIG. 1(b) is a graph showing an example of the temperature dependence of the peak value of the ODMR spectrum. As shown in the graph G1 of FIG. 1(a), when a microwave with a frequency F of about 2.87 GHz is irradiated, the amount of fluorescence attenuates by about 0.03. The peak (minimum value) is divided into two due to the effect of crystal distortion. Each of the first frequency F1 and the second frequency F2 indicates a frequency at which the amount of fluorescence becomes a minimum value. The second frequency F2 is higher than the first frequency F1. The horizontal axis of the graph shown in FIG. 1(b) indicates the temperature T, and the vertical axis indicates the frequency of the ODMR spectrum peak. The temperature T indicates the ambient temperature of the inorganic fluorescent particles. The graph G2 shows the change in the second frequency F2 with respect to the change in temperature T. The graph G3 shows the change in the first frequency F1 with respect to the change in temperature T. As shown in the graphs G2 and G3, it can be seen that the ODMR spectrum peak shifts due to the change in the ambient temperature. Therefore, the temperature can be measured based on this peak shift.

[0022] 2. Process (a) In the step (a), a target object containing inorganic fluorescent particles is irradiated with a plurality of types of microwaves having mutually different frequencies.

[0023] Inorganic fluorescent particles are not particularly limited as long as they have electron spin activity.Specific examples include diamond, silicon carbide, zinc oxide, two-dimensional materials (e.g., hexagonal boron nitride, etc.).Of these, diamond (especially, nano-sized (average particle size is less than 1000 nm) diamond (nanodiamond)) is preferred.

[0024] The diamond may be either single crystal or polycrystalline. Examples of synthetic diamond include diamonds synthesized by CVD, high temperature and high pressure, explosion, etc. Examples of diamond include I-type and II-type (IIa-type, Iib-type, etc.) diamonds.

[0025] The shape of diamond is not particularly limited. Examples of the shape include particles, thin films, sheets, etc. The size varies depending on the shape, but in the case of particles, the average particle diameter can be, for example, 1 nm to 500 nm. The average particle diameter is preferably about 10 to 200 nm, more preferably about 30 to 150 nm.

[0026] The diamond is preferably a diamond containing an NV center (a light-emitting center formed by bonding nitrogen atoms contained as impurities with vacancies lacking carbon atoms in an appropriate positional relationship) (a diamond containing an NV center). The NV center may be naturally occurring or may be artificially introduced. The method of artificially introducing the NV center is not particularly limited, and examples thereof include a method of injecting nitrogen atoms and then annealing, and a method of introducing nitrogen atoms during chemical vapor deposition (CVD) of diamond.

[0027] Diamond can also be surface-modified. The method of surface modification is not particularly limited, but for example, diamond can be treated under strong oxidation conditions as necessary to convert the carbon on the surface into a carboxyl group, or diamond can be reduced to introduce a hydroxyl group, or other functional groups (amino group, thiol group, etc.) can be introduced according to or in accordance with a known method, and various molecules or substances can be linked through them to perform surface modification. Surface-modifying molecules are not particularly limited, and include, for example, water-soluble polymers such as polyglycerol and polyethylene glycerol, various low molecular weight compounds such as proteins, peptides, nucleic acids, and pharmaceutical compounds.

[0028] The inorganic fluorescent particles may be of one type alone or in combination of two or more types.

[0029] The object is a subject of temperature measurement, and is not particularly limited. The object is preferably a cell, a microorganism, an organoid, etc. The inside of these measurement objects is a dynamic environment, and therefore, when performing real-time temperature measurement, the number of photons measured fluctuates, etc., which causes artifacts in the temperature measurement value. According to the measurement method of the present invention, even for such an object, it is possible to measure the temperature with higher accuracy.

[0030] The cells are not particularly limited, and examples thereof include vascular endothelial cells, endothelial progenitor cells, stem cells (e.g., bone marrow-derived stem cells, adipose tissue-derived stem cells, mesenchymal stem cells, pluripotent stem cells (iPS cells, ES cells, etc.), etc.), muscle cells (skeletal muscle cells, smooth muscle cells, cardiac muscle cells), muscle precursor cells (e.g., cardiac precursor cells, myoblasts, etc.), immune cells (T cells, etc.), nerve cells, etc.

[0031] Microorganisms are not particularly limited as long as they are organisms that cannot be seen with the naked eye, or organisms that can be seen with the naked eye but whose structure cannot be distinguished, and examples of such organisms include bacteria, single-celled organisms, plankton, larvae, nematodes, etc.

[0032] Organoids include brain organoids, cerebellar organoids, inner ear organoids, thyroid organoids, thymus organoids, testicular organoids, liver organoids, spleen organoids, intestinal organoids, epithelial organoids, lung organoids, kidney organoids, embryos, and the like.

[0033] In the case of an object containing inorganic fluorescent particles, the inorganic fluorescent particles may be present inside the object or may be attached to the outside, and the former is preferable. In the former case, the influence of measurement noise due to the environment inside the object is greater, but the measurement method of the present invention makes it possible to measure the temperature with higher accuracy even in this case. Objects containing inorganic fluorescent particles can be obtained by various methods. For example, when the object is a cell, the inorganic fluorescent particles can be brought into contact with the cell to cause the cell to take up the inorganic fluorescent particles.

[0034] The amount of inorganic fluorescent particles in the subject can be appropriately determined depending on the type of subject, the type of inorganic fluorescent particles, etc. For example, in the case of a nematode with a body length of about 1 mm, the amount of inorganic fluorescent particles introduced into one nematode is, for example, 1 to 100 ng.

[0035] The object containing the inorganic fluorescent particles is arranged so that the excitation light irradiation, the micro light irradiation, and the fluorescence collection can be performed to measure the temperature of the present invention. Specifically, the object is arranged on the sample stage on the objective lens in the apparatus shown in FIG.

[0036] In the measurement method of the present invention, the microwaves irradiated are a plurality of types of microwaves having mutually different frequencies.

[0037] The frequency of the microwave is usually 9 GHz or less, and is selected from a frequency range that shows a linear approximation on both sides of the expected ODMR spectrum peak (see FIG. 2). The frequency range is desirably determined in advance before measurement according to the type of inorganic fluorescent particles and the target object. The type of microwave (frequency) is usually an even number, and from the viewpoint of measurement accuracy, measurement efficiency, etc., it is preferably 2 to 10 types, more preferably 4 to 10 types, further preferably 6 to 8 types, and particularly preferably 6 types. It is desirably set so that the measured fluorescence amount is approximately the same as the fluorescence amount measured at the corresponding microwave frequency on the opposite side of the peak (see FIG. 2). That is, in FIG. 2, it is desirably designed so that the measured fluorescence intensity (I1) is approximately the same as the fluorescence intensity (l6) measured at the corresponding frequency (f6). The frequency difference between each microwave on one side of the ODMR spectrum peak (for example, X in FIG. 2) is preferably 1 to 5 MHz.

[0038] The irradiation time for each microwave of each frequency is not particularly limited, but is, for example, 10 μs to 1000 μs, preferably 30 to 300 μs, and more preferably 50 to 200 μs, from the viewpoint of measurement accuracy, measurement efficiency, etc. It is preferable that the irradiation times for each microwave are approximately the same, and for example, the longest irradiation time relative to the shortest irradiation time is, for example, 200% or less, 150% or less, 120% or less, or 110% or less.

[0039] Microwaves of each frequency are usually irradiated repeatedly. For example, in the example of FIG. 2, microwaves are irradiated in the order of frequency f1, frequency f2, frequency f3, frequency f4, frequency f5, and frequency f6, and then microwaves are irradiated in the order of frequency f1, frequency f2, frequency f3, frequency f4, frequency f5, and frequency f6, and this cycle is repeated. In addition, by continuing this repetition for a certain period of time (for example, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 60 minutes or more, 2 hours or more, 5 hours or more, 8 hours or more), the change in temperature of the object over time can be measured.

[0040] Microwave irradiation is performed using a suitable microwave source. Repeated irradiation of microwaves of each frequency can be performed, for example, by preparing multiple microwave sources of each frequency, connecting them to a switching device, and operating the device to switch the microwave sources sequentially at a predetermined time. In addition, the microwaves generated from the microwave source are usually amplified through an amplifier before being irradiated to the object.

[0041] In step (a), a stimulus can also be applied to the object containing inorganic fluorescent particles. When the object is a cell, a microorganism, an organoid, etc., if a temperature change occurs due to the application of a stimulus, the temperature change can be measured. The type of stimulus is not particularly limited, and examples thereof include changes in culture conditions (e.g., temperature, pH, light conditions, etc.), addition of a test substance, etc. The test substance is not particularly limited, and examples thereof include antibodies, proteins, nucleic acids, physiologically active substances, vesicles, bacteria, viruses, polypeptides, haptens, therapeutic drugs, metabolites of therapeutic drugs, etc.

[0042] 3. Process (b) In step (b), the fluorescence intensities of the inorganic fluorescent particles when irradiated with various types of microwaves are measured using separate photon counters.

[0043] The fluorescence intensity of inorganic fluorescent particles during microwave irradiation is usually measured by irradiating microwaves under the condition that the inorganic fluorescent particles are continuously irradiated with the excitation light of the particles, and measuring the intensity (I1, I2...) of the fluorescence (fluorescence L1, L2...) at the time of irradiation of the microwave (frequency f1, f2...). The wavelength of the excitation light varies depending on the type of inorganic fluorescent particles, and can be set appropriately. For example, when using NV center-containing diamond, the wavelength of the excitation light is, for example, 490 to 580 nm, preferably 520 to 560 nm. The wavelength of the fluorescence also varies depending on the type of inorganic fluorescent particles. For example, when using NV center-containing diamond, the wavelength of the fluorescence is, for example, 637 to 800 nm. Irradiation with excitation light and detection of fluorescence may be performed, for example, as follows: a continuous wave laser of typical excitation intensity is used for excitation, a microscope objective is used for both excitation and fluorescence collection, the fluorescence is extracted (e.g., by a splitter such as a dichroic beam splitter or a filter such as a long pass filter), and, if necessary, the fluorescence is coupled into an optical fiber acting as a pinhole or a pinhole is used to detect the fluorescence with a photodiode such as an avalanche photodiode or other photodetector.

[0044] The fluorescence intensity of inorganic fluorescent particles when irradiated with various microwaves is measured by separate photon counters. That is, the intensity of the fluorescence (fluorescence L1, L2...) at the time of irradiation with various microwaves (frequencies f1, f2...) is measured by separate photon counters (counter 1 for the intensity of fluorescence L1, counter 2... for the intensity of fluorescence L2). There is no particular limitation on the photon counter, and various counters can be used. As the photon counter, a single counter in one independent measuring device may be used, or multiple counters in the measuring device may be used. Also, multiple measuring devices may be used in combination to prepare the required number of counters (= the number of types of microwaves). The fluorescence intensity (I1, I2...) of inorganic fluorescent particles when irradiated with various microwaves can be obtained by measuring with separate photon counters (counter 1 for the intensity of fluorescence L1, counter 2... for the intensity of fluorescence L2).

[0045] The fluorescence intensity may be an absolute value or a relative value.

[0046] The inorganic fluorescent particle to be measured is usually one particle, but it is also possible to measure multiple particles in parallel. If the inorganic fluorescent particle moves during measurement, the inorganic fluorescent particle can be appropriately tracked to continue tracking the particle to be measured, and the temperature change over time can be measured with higher accuracy. The tracking method is not particularly limited, and the particle can be tracked using a known tracking technique.

[0047] 4. Process (c) In step (c), the fluorescence intensity is corrected based on the error in the number of pulses counted between the photon counters.

[0048] The inventors discovered that there exists a difference in photocounting response (error in the number of pulses derived from photons) between multiple photon counters, which causes artifacts in temperature measurements when performing real-time temperature measurements in dynamic environments such as at the cellular or individual level. Therefore, by correcting this error, it becomes possible to perform temperature measurements with higher accuracy.

[0049] It is desirable to measure the error in the number of pulses measured between the photon counters in advance. The measurement of the error is not particularly limited, but can be performed, for example, as follows. A single inorganic fluorescent particle is irradiated with a plurality of microwaves of frequencies used in temperature measurement, each of a plurality of laser intensities (e.g., 3 to 20, 4 to 15, 6 to 12) that are gradually increased, and the number of photons (p1, p2...) for the fluorescence (fluorescence L1, L2...) at the time of irradiation with each microwave (frequency f1, f2...) is measured using separate photon counters (counter 1 is the number of photons for fluorescence L1, and counter 2... is the number of photons for fluorescence L2). Based on the measured values, the measurement error (difference in the number of photons measured under the same conditions) between the two corresponding photon counters is calculated. The "two corresponding photon counters" refer to two counters (counter 1 corresponds to counter 6, counter 2 corresponds to counter 5, and counter 3 corresponds to counter 4) that measure corresponding frequencies on both sides of the peak of the ODMR spectrum assumed during temperature measurement (see FIG. 2; f1 corresponds to f6, f2 corresponds to f5, and f3 corresponds to f4). The graph showing the measurement error as shown in FIG. 4 can be obtained by performing, for example, polynomial fitting based on the above measurement values.

[0050] The fluorescence intensity obtained in step (b) is corrected based on the error. The method of correction is not particularly limited, but for example, a correction value (correction values ​​c1, c2, etc.) can be obtained by subtracting an error from the measured value of either one of the two corresponding fluorescence intensities (see FIG. 2; I1 and I6 correspond to each other, I2 and I5 correspond to each other, and I3 and I4 correspond to each other) or by adding an error to the measured value of either one of the fluorescence intensities.

[0051] Specifically, step (c) includes, for example, a step of subtracting a previously measured value of the error in the number of pulses counted between the photon counters from the measured value of either one of the two corresponding fluorescence intensities, or adding the value to the measured value of either one of the two corresponding fluorescence intensities.

[0052] 5.Process (d) In step (d), the temperature of the object is calculated based on the obtained correction value.

[0053] The calculation of the temperature of the object is not particularly limited, and can be performed according to or in accordance with a known method. When four types of microwaves are used, the temperature can be calculated, for example, according to the method described in Patent Document 1. When six types of microwaves are used, for example, from six correction values ​​corresponding to each of the six frequencies, the temperature can be calculated for each of two combinations (a combination of c1-c6 and c2-c5, a combination of c2-c5 and c3-c4, and a combination of c1-c6 and c3-c4) in which two corresponding correction values ​​(c1-c6, c2-c5, c3-c4) have approximately the same value, for example, according to the method described in Patent Document 1, and the average value can be used as the final measured value. Specifically, the temperature can be calculated, for example, according to the method and formula described in the later-described Examples ("1. Temperature Measurement Method").

[0054] When six types of microwaves are used, the step (d) specifically involves, for example, calculating the correction value obtained in the step (c) according to the following formula:

[0055]

number

[0056] [In the formula, α indicates the temperature dependence of the luminescence center (NV). δω indicates the frequency difference between the first and third, or the fourth and sixth, frequencies from the low frequency side of the microwave. I1 to I6 indicate the correction values ​​obtained by irradiating six types of microwaves, respectively.] Substituting the temperature change at the luminescence center (δT NV )

[0057] Also, for temperature, it is possible to measure with higher accuracy by taking the average value over a certain period of time. Microwaves of each frequency are usually repeatedly irradiated (for example, in the example of FIG. 2, microwaves are irradiated in the order of frequency f1, frequency f2, frequency f3, frequency f4, frequency f5, and frequency f6, and then microwaves are irradiated in the order of frequency f1, frequency f2, frequency f3, frequency f4, frequency f5, and frequency f6, and this cycle is repeated). However, for example, the average (for example, adjacent average, moving average, etc.) of temperatures calculated from each cycle within a certain period of time (for example, 0.1 to 180 seconds, 0.3 to 120 seconds, 1 to 100 seconds, 3 to 100 seconds, 10 to 80 seconds, 20 to 50 seconds) can be used as the temperature measurement value.

[0058] By utilizing the above-mentioned technology, it may be possible to measure nanoscale thermal events with high precision in real time, measure individual metabolism, test the effectiveness of health foods such as fat burning, and measure the temperature of metabolic changes caused by drugs.

[0059] The measurement method of the present invention can be carried out using a temperature measuring device (sometimes referred to as the "measurement device of the present invention" in this specification) that includes (A) a microwave irradiation device, (B) a photon counter, (C) a calculation unit that corrects fluorescence intensity, and (D) a calculation unit that calculates temperature.

[0060] The microwave irradiation device and the photon counter are as described above.

[0061] The calculation unit that corrects the fluorescence intensity and the calculation unit that calculates the temperature may be one calculation unit or may be separate calculation units.

[0062] The calculation unit that corrects the fluorescence intensity obtains information about the fluorescence intensity measured by the photon counters and performs correction based on the error in the number of pulses measured between the photon counters. The calculation unit that calculates the temperature obtains information about the obtained correction value and calculates the temperature. The processing content in these calculation units is as described above and is executed by a computer program stored in advance.

[0063] The measuring device of the present invention preferably further comprises a particle tracking system. As the system, a system utilizing a known particle tracking technology (such as a piezo stage) can be used.

[0064] The measuring device of the present invention may further include a sample stage for placing an object containing inorganic fluorescent particles, a microscope objective lens, a fluorescent irradiation device, a display unit for displaying calculated temperature information, etc., and may be an all-in-one device that can perform the measuring method of the present invention by itself. EXAMPLES

[0065] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0066] 1.Temperature measurement method The ODMR temperature measurement method in the examples is described below. A schematic diagram of the apparatus is shown in Figure 3. For excitation, a typical excitation intensity of ca. 5 kW cm was used. -2A continuous wave 532 nm laser was used. A numerical aperture 1.4 oil immersion microscope objective was used for both excitation and fluorescence collection. The NV fluorescence was filtered by a dichroic beam splitter (Semrock, FF560-FDi01) and a long pass filter (Semrock, BLP01-561R, or BLP01-635R-25) to remove residual green laser scattering. The fluorescence was then coupled into an optical fiber (Thorlabs, 1550HP, core diameter approx. 10 μm) which acted as a pinhole. The fiber-coupled fluorescence was finally detected by an avalanche photodiode (APD, Perkin Elmer SPCM AQRH-14). The sample was mounted on a piezo stage allowing raster scanning and particle tracking. The APD output was fed into two data acquisition boards, each with four pulse counters (DAQ-1 BNC, National Instruments) and two pulse counters (DAQ-2 BNC, National Instruments). All photon counting measurements except the six-point measurements were performed by a DAQ board (USB-6343 BNC). For fluorescence spectral measurements, a spectrometer (Princeton, LNCCD) equipped with a liquid nitrogen-cooled charge-coupled device camera was used. Spectral measurements and particle tracking were performed simultaneously by inserting a fiber pigtail beam splitting system into the optical fiber line to prevent chromatic aberrations caused by particle motion.

[0067] To perform both CW and multipoint ODMR measurements, one free-standing microwave source (Rohde&Schwarz, SMB100A) and five USB-powered microwave sources (USG-LF44, Texio) were connected to an SP6T switch (General Microwave, F9160) with a switching time of 250 ns. It was then amplified (Minicircuits, ZHL-16W-43+) and sent to a microwave linear antenna placed on a cover slip (25 μm thin copper wire) and sealed with a cell culture dish with a hole in the center. The typical microwave excitation power is estimated to be 5 [A / m] in terms of magnetic field strength, taking into account the input and transmission power of the antenna, and the electromagnetic field simulation by the finite element method (COMSOL). For CW-ODMR measurements, the APD detection common to both gates was gated on and off the microwave irradiation using the SP6T switch and a bit pattern generator (Spincore, PBESR-PRO-300) with a gate width of 200 μs. This was followed by a 100 μs laser shutoff period and I PL ON and I PL OFF In the example, no external magnetic field was applied. In the multipoint ODMR measurements, the APD detection was gated for each microwave frequency with a gate width of 100 μs common to all six gates, followed by a 5 μs interval. The obtained photon counts at the six frequencies were input into the following equation to obtain the temperature estimate of the NV center (T NV ) was calculated.

[0068]

number

[0069] [where α is the temperature dependence of the NV center, -74 kHz °C -1 It is.]

[0070] 2. Six-point measurement and ODMR peak shift due to temperature change To realize nanodiamond quantum thermometry in vivo, we developed a real-time in vivo temperature measurement system based on a confocal fluorescence microscope with fast particle tracking capability and a high-precision temperature estimation protocol (see "1. Temperature measurement method" above and Figure 3). In particle tracking, the system measures the fluorescence intensity along the x, y and z directions and moves the focus back to the respective fluorescence maximum each time. The repositioning typically takes 2.8 seconds and is repeated every 4 seconds.

[0071] High-precision quantum thermometry is based on the detection of temperature-dependent peak shifts of the optically detected magnetic resonance (ODMR) line of the nitrogen vacancy (NV) center of NDs. In particular, we adopted a multipoint ODMR measurement protocol, in which the fluorescence intensity is measured at six frequencies symmetrically located with respect to the ODMR peak. The fluorescence intensity at four of the six frequency points provides three sets of temperature estimates following the formula given in “1. Temperature measurement method” above, and finally, the temperature estimate can be given by taking their average. Experimentally, six frequencies are output sequentially from a frequency selector with a pulse width of 100 μs and an interval of 5 μs. These time-controlled microwave pulse trains are sent to a microwave antenna fabricated on a cell culture dish. NDs or ND-labeled C. elegans worms are placed on the culture dish.

[0072] The advantage of choosing six points instead of four points as previously reported (Patent Document 1) is that it improves temperature precision: compared to choosing four points with the same photon flux, six-point analysis uses ⅔ the number of photons per second while performing four-point analysis in triplicate, thus reducing measurement noise (Equation below).

[0073]

number

[0074] In practice, increasing the number of frequency points also provides more information about the ODMR spectral shape, which is useful for detailed analysis of the temperature profile. In this study, a moving average was used; however, more advanced data estimation such as Kalman filtering can also be effective.

[0075] We found that to realize real-time monitoring in a dynamic environment, it is important to calibrate the photon count responsivity of each pulse counter. Each counter was found to have a very small photoresponsivity difference of <5% (see Figure 4). Figure 4a shows the counter values ​​of NV fluorescence at six frequencies (I1-I6) as a function of excitation laser power. Ideally, I1, I2, and I3 should show the same responsivity slope as their corresponding I4, I5, and I6. Figure 4b shows their differences (I1-I6, I2-I5, and I3-I4) as a function of the photon number of either I1, I2, or I3, respectively. Therefore, in the following tests, we performed counter calibration to correct this deviation (error).

[0076] We first performed temperature measurements on NDs attached to a cover glass (see Fig. 3). As shown in Fig. 5a, step changes in the photon count caused artifacts in the temperature estimate of the NV center, even though the actual temperature did not change. By calibrating I1–I6 (I2–I5 and I3–I4) over the full range of photon counts used in the experiment, we were able to significantly suppress the noise and drift. With these counter calibrations, the temperature measurements were no longer affected by photon count variations.

[0077] Next, the microscope objective lens (T OBJ The temperature measurement specifications (precision, accuracy, time resolution, and stability) were evaluated by stepping the temperature of the sample (T) from 20 to 40 °C (Fig. 5b). It started at 44.0 °C and was then reduced to 40.2 °C. The temperature was then increased slightly to 40.9 °C in 40.7 min. Importantly, the T NVcould clearly detect this small temperature change of only 0.7 °C, as shown in the inset of Fig. 5b. This temperature transition occurred with a characteristic time constant of 19 s in a practical Boltzmann fit and was completed in about 1.5 min. T OBJ and T NV The time difference between was 1.2 minutes, demonstrating that the nanodiamond thermometer can detect transient temperature changes of less than 1°C in real time.

[0078] In the low temperature range below 35°C, T NV and T OBJ This difference is due to the fact that OBJ This has nothing to do with the accuracy of the thermometer, since there is not enough waiting time for the temperature to fully thermalize. Rather, it accurately reflects the actual temperature of the surface of the cover glass, since the heat dissipation is proportional to the difference with room temperature according to Fourier's law. It takes more than an hour to reach full thermal equilibrium.

[0079] Heating a microscope objective lens causes more significant fluctuations and drift in focal position than lowering the temperature. Therefore, to demonstrate the robustness of the thermometer, we turned the heater off at 207 min and then turned it on at 218 min to 35 °C. During this rapid thermal event, the system was able to track the ND position while showing a correct temperature estimate. From a stepwise change in the range of 35–45 °C, the accuracy of the current temperature measurement was determined to be 0.16 °C by an integration time of 34 s, and the precision was less than 0.3 degrees root-mean-square (Figure 5c). Thus, the sensitivity was 1.6 °C / √Hz. Such a small net precision in real time is a remarkable feature of the current nanoscale thermometer, which is an important milestone for biological in vivo applications.

[0080] 3. Temperature Measurement in Living C. elegans Having established a robust and accurate temperature measurement that works in real time, we tested local temperature monitoring in live worms. The NDs used to label worms were highly water-soluble nanodiamonds with a polyglycerol coating on their surface. We introduced these NDs into worms by microinjection into the gonads and cultured them overnight to allow the NDs to be incorporated into the cells. The labeled worms were anesthetized and transferred to the vicinity of the microwave antenna in an antenna-integrated culture dish. They were sandwiched between an agar pad and a cover glass filled with buffer.

[0081] Figure 6a-d show the DIC (differential interference contrast), green, red confocal fluorescence, and their composite images, respectively, of a labeled worm. The NDs are distributed throughout the entire area in the image, allowing us to measure the ODMR signals of different parts of the worm. In living worms, the NDs move continuously during temperature observations, with a typical speed of around 200 nm / min measured by time-lapse images. In the following experiments, we continued to track these NDs while measuring the local temperature.

[0082] Figure 7a-b show merged micrographs and cw-ODMR spectra of a single ND, indicated by an arrow near an embryo. ODMR contrast is generally weaker in worms due to background fluorescence. Typically, ODMR contrasts of 0.9-0.94 are obtained in worms compared to 0.88 on coverslips. From this, it was estimated that the intensity of the background fluorescence was 0.6-1.0 times that of the ND fluorescence. Figure 7c shows the T over 1 h during the course of temperature changes with a microscope-objective heater. NV FIG. 1 shows the temporal profile of T OBJ was initially set to 34.0°C (RT = 21.5°C at this point). At 5 minutes, the heater was turned off and T NVwas gradually decreased towards RT. At 38.7 min, the heater was turned on and adjusted to 29.5 °C. The measured profile of TNV (red) was in excellent agreement with TOBJ (blue). The precision and accuracy of this in vivo measurement were ±0.39 °C (integration time 34 s) and <0.6 °C, respectively (Fig. 7e).

[0083] 4. Detection of thermogenic response to drug stimulation in C. elegans To demonstrate the applicability of the present thermometry to in vivo heat generation research, we measured the internal temperature of the worm when it was stimulated by FCCP (carbonyl cyanide-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler. Figure 8a shows the temporal profile of the internal temperature of the worm when it was stimulated by FCCP. After the start of the measurement, T NV The T decreased continuously, which was actually a baseline drift, which was frequently observed in vivo. After 6 min, a small amount of 60 μM FCCP solution was added to the medium. Between 10 and 18 min, the T NV The baseline drift of was shifted down sharply with short transition times of 4 and 12 s in the drop and recovery, respectively. This was mainly due to the tracking system miscapturing a nearby ND with a slightly different ODMR peak (see Fig. 8b). At 24 min (18 min after FCCP addition), the temperature gradually increased, reaching a maximum of about 3 °C at 40–50 min. Between 25 and 35 min, there was indeed a strong fluctuation in the number of photons, which was reflected in the temperature profile. During this period, the ND position fluctuated greatly because the worm moved. Indeed, the embryo located at the bottom of the image moved after this period (Fig. 8b). However, our system was able to track the ND with the temperature information. Only for 2 min between 28 and 30 min, the ND moved beyond the adjustable range of tracking and we lost the ND position. However, we could find the same ND by wide-field fluorescence imaging and place the ND back in focus, which was consistent with the T NV Between 60 and 65 minutes, the exothermic reaction appeared to be complete. After 65 minutes, T NVThe baseline drift of NDs seemed to increase in sync with the decrease in the number of photons. It is known that the increase in temperature promotes non-radiative relaxation due to heat, and the fluorescence of NDs decreases. Conversely, the amount of fluorescence increases with a decrease in temperature. From this point of view, the fluorescence intensity and temperature information are inversely correlated. However, since the fluorescence intensity is also affected by the time-dependent changes in the transmittance and refractive index of the biological sample, the correlation is not necessarily observed in some cases.

Claims

1. 1. A method for measuring the temperature of an object based on optically detected magnetic resonance of inorganic fluorescent particles, comprising: (a) irradiating an object containing inorganic fluorescent particles with a plurality of microwaves having different frequencies; (b) measuring the fluorescence intensity of the inorganic fluorescent particles when irradiated with various microwaves using separate photon counters; (c) correcting the fluorescence intensity based on the error in the number of pulses counted between the photon counters; and (d) calculating the temperature of the object based on the obtained correction value; The method includes:

2. The method of claim 1 , wherein the inorganic fluorescent particles are NV center-containing diamond.

3. The method according to claim 1 or 2, wherein the plurality of types of microwaves is 2 to 10 types.

4. The method for measuring a change in temperature of an object over time, wherein the object is a cell, a microorganism, or an organoid, according to any one of claims 1 to 3.

5. 5. The method according to claim 1, wherein the step (c) comprises a step of subtracting a previously measured value of an error in the number of pulses counted between the photon counters from the measured value of either one of the two corresponding fluorescence intensities, or adding the previously measured value of the error in the number of pulses counted between the photon counters to the measured value of either one of the two corresponding fluorescence intensities.

6. The plurality of types of microwaves are six types; and The step (d) calculates the correction value obtained in the step (c) by the following formula: [0010] [In the formula, α indicates the temperature dependence of the luminescence center (NV). δω indicates the frequency difference between the first and third, or the fourth and sixth, microwave frequencies from the low frequency side. I 1 ~I 6 indicates the corrected values ​​obtained for each of the six types of microwave irradiation.] and the temperature change at the luminescence center (δT NV The method according to any one of claims 1 to 5, further comprising the step of calculating

7. A temperature measuring device that measures the temperature of an object based on the optically detected magnetic resonance of inorganic fluorescent particles, comprising: (A) a microwave irradiation device that irradiates an object containing inorganic fluorescent particles with multiple types of microwaves having different frequencies; (B) a photon counter that separately measures the fluorescence intensity of the inorganic fluorescent particles when irradiated with each type of microwave; (C) a calculation unit that corrects the fluorescence intensity based on the error in the number of pulses measured between the photon counters; and (D) a calculation unit that calculates the temperature.

8. The temperature measurement device of claim 7 , further comprising: (E) a particle tracking system.

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