JP7371888B2 - Carbon isotope analysis device for plant sample analysis and carbon isotope analysis method for plant sample analysis using the same - Google Patents

Description

The present invention relates to a carbon isotope analysis device for plant sample analysis and a carbon isotope analysis method for plant sample analysis using the same.

Carbon isotopes have been used in a wide range of applications across the humanities and sciences, including environmental dynamic assessments based on the carbon cycle and historical empirical research using dating. Carbon isotopes vary slightly depending on region and environment, but the stable isotopes ^12C and ^13C are 98.89% and 1.11%, respectively, and the radioactive isotope ^14C is 1x10 ^-10 % naturally occurring. exists in Isotopes differ only in weight, but chemically they behave in the same way. Therefore, by artificially increasing the concentration of isotopes with a low abundance ratio and performing accurate measurements, it is possible to investigate various reaction processes. Observation becomes possible.

In addition to applications in medicine and pharmacy, ¹⁴ C tracers are used as useful tools for evaluating various dynamics important in plant physiology in plants, particularly in crops such as grains and vegetables. For example, a research group led by M. Calvin et al. at the University of California in the United States caused plants to absorb ¹⁴ CO ₂ through photosynthesis and elucidated the scientific reaction process (see, for example, Non-Patent Document 1). The process elucidated through this research is called the Calvin Cycle, and was awarded the Nobel Prize in Chemistry in 1961.
In such applications of ¹⁴ C tracers in plant physiology, methods based on radiation measurement have basically been used. For example, a research group led by Tanoi et al. at the University of Tokyo has developed a system that images (live imaging) ¹⁴ C-derived beta rays administered to plants without stopping their biological activities (for example, non-patent document 2). reference.).
On the other hand, while the imaging method using an imaging plate is a powerful means for visually evaluating dynamics, the β-ray energy from ¹⁴ C is weak and is absorbed within the plant body, resulting in low measurement sensitivity. Quantitative analysis is difficult.

Conventionally proposed ^14C analysis methods include liquid scintillation counting (hereinafter also referred to as "LSC") and accelerator mass spectrometry (hereinafter also referred to as "AMS"). Can be mentioned.

Patent No. 3390755

“M. Calvin, “The path of carbon in photosynthesis”, Science, 135, 879-889 (1962).” “R. Sugita, et al., “Visualization of uptake of mineral elements and the dynamics of photosynthates in arabidopsis by a newly developed real-time radioisotope imaging system (RRIS)”, Plant Cell Physiol., 57, 743-753 (2016 ).” "I.Galli et al.,Phy. Rev. Lett. 107, 270802 (2011)"

Since LSC is a relatively small device of table top size, simple and rapid analysis is possible. However, when measuring a plant sample with LSC, if the plant sample is placed in an organic solvent without pretreatment, the solution becomes colored and the number of scintillation light decreases (color quenching). Furthermore, the detection limit concentration of ¹⁴ C in LSC is as high as 10 dpm/mL, which limits detection sensitivity, making it difficult to measure with a small amount of sample.

On the other hand, AMS has a detection limit concentration of ¹⁴ C as low as 0.001 dpm/mL, which is more than 1000 times lower than LSC's detection limit concentration of ¹⁴ C, so it has high detection sensitivity and can be used, but the device is large and expensive. Therefore, its use was restricted. Several techniques have been proposed as means to solve this AMS problem (for example, see Non-Patent Document 3 and Patent Document 1), but Cavity Ring-Down Spectroscopy (hereinafter referred to as "CRDS") Although ^{the 14} C analysis was demonstrated using the 4.5 μm band laser beam generator used, it had an extremely complicated structure, so a simpler and more user-friendly ¹⁴ C analysis device and method were needed. It was getting worse. Also, the structure was not designed with analysis of plant samples in mind.
Based on the above, there was a need for a carbon isotope analyzer suitable for analyzing plant samples and a carbon isotope analysis method using the same, which has a simple structure but can perform sensitive analysis without complicated pretreatment. .

The present invention relates to the following contents.
[1] It has a ¹⁴ CO ₂ photosynthesis labeling system for plant samples and a carbon isotope analyzer for analyzing plant samples, and the carbon isotope analyzer for analyzing plant samples detects gas containing ¹⁴ C to ¹⁴ CO ₂ a carbon dioxide isotope generating device including a combustion unit that generates a carbon dioxide isotope, a light generating device, an optical resonator having a pair of mirrors, and a spectroscopic device including a photodetector that detects the intensity of transmitted light from the optical resonator. , a vial for generating 14 CO 2 gas, a sample bag for accumulating the generated gas, a desiccator into which the plant sample is introduced, a circulation pump, a first pipe connecting the vial and the sample bag, and a ^sample bag _. It has a second pipe that connects the bag and the desiccator, and a third pipe that connects the sample bag and the desiccator via a circulation pump, and the volume of the sample bag is small compared to the volume of the desiccator. ¹⁴ CO ₂ gas generated in the vial is accumulated in the sample bag by opening only the first pipe from the first pipe to the third pipe, and then opening only the second pipe from the first pipe to the third pipe and placing it in the desiccator. For plant sample analysis, the chamber is filled with ¹⁴ CO ₂ gas, and then the second and third pipes from the first pipe to the third pipe are opened and the gas is circulated between the sample bag and the desiccator using a circulation pump. system.
[2] The plant sample analysis according to [1] , wherein the combustion section includes a combustion furnace arranged substantially perpendicular to the direction of gravity, a sample boat, a carrier gas supply tank, and a dehumidifier. System for.
[3] The plant sample according to [1] or [2] , further comprising a carbon dioxide isotope trap equipped with a cooling device for freezing ¹⁴ CO ₂ , disposed between the carbon dioxide isotope generating device and the spectroscopic device. Analytical system.
[4] The light generating device includes a light source, an optical switch that controls on/off of the light from the light source, and a mirror that reflects the light from the optical switch and sends the light back to the optical switch, [1] to [3 ] ] The plant sample analysis system according to any one of the above.
[5] The plant sample analysis system according to [4] , wherein the optical switch is an acousto-optic modulator.
[6] The light generating device includes a branching means for branching the light from the light source, a condensing lens for condensing the light from the branching means, and a condensing lens for reflecting the light from the condensing lens and passing it through the condensing lens and the branching means. The plant sample analysis system according to [4] or [5] , comprising a mirror that sends light back to the light source.
[7] The plant sample analysis system according to any one of [4] to [6], wherein the light source generates optical frequency comb light.
[8] The plant sample analysis system according to any one of [4] to [7], wherein the light source is a fiber laser.
[9] The plant sample analysis system according to any one of [4] to [6], wherein the light source is a mid-infrared quantum cascade laser.
[10] The light source includes a mid-infrared quantum cascade laser as the main light source, an optical comb source that generates an optical comb consisting of a bundle of light with a narrow line width in the frequency range of 4500 nm to 4800 nm, and a main light source. A beat signal measuring device according to any one of [4] to [6] , comprising a photodetector that measures a beat signal caused by a frequency difference between the light from the optical comb source and the light from the optical comb source. System for plant sample analysis.
[11] A step of photosynthesizing the plant sample using ^{a 14} CO ₂ photosynthesis labeling method of the plant sample and labeling the plant sample with ¹⁴ C, and a step of generating ¹⁴ CO ₂ from the ¹⁴ C in the plant sample; A step of filling CO ₂ into an optical resonator, a step of irradiating the optical resonator with irradiation light having an absorption wavelength for ¹⁴ CO ₂ , and a step of irradiating ¹⁴ CO ₂ with irradiation light and causing it to resonate. The ¹⁴ CO ₂ photosynthesis labeling method for plant samples includes the steps of measuring the intensity of transmitted light and calculating ^{the 14} C concentration from the intensity of the transmitted light, and includes the steps of generating ¹⁴ CO ₂ gas in a vial. a step of accumulating the generated 14 CO 2 gas in a sample bag; a step of filling a desiccator into which the plant sample is introduced and which has a larger volume than the sample bag with 14 CO ² gas _from the ^sample bag _; A carbon isotope analysis method for plant sample analysis , comprising the step of circulating gas between a bag and a desiccator .
[12] Between the process of irradiating the irradiation light and the process of measuring the intensity of the transmitted light, the light from the light source is introduced into the optical switch, and the light emitted from the optical switch is sent back to the optical switch to turn the light on and off. The carbon isotope analysis method for plant sample analysis according to [11] , further comprising a controlling step.
[13] As irradiation light, a plurality of lights are passed through a nonlinear optical crystal to generate an optical comb with a mid-infrared optical frequency in the wavelength band of 4.5 μm to 4.8 μm from the difference in frequency, [11] or [ 12] carbon isotope analysis method for plant sample analysis.

According to the present invention, there is provided a carbon isotope analyzer suitable for analyzing plant samples and a carbon isotope analysis method using the same, which has a simple structure and can be analyzed with high sensitivity without complicated pretreatment. be done. Furthermore, a system for analyzing plant samples is provided, which includes the above carbon isotope analyzer and a ¹⁴ CO ₂ photosynthesis labeling system.

FIG. 1 is a conceptual diagram of a first embodiment of a carbon isotope analyzer. FIG. 2 is a conceptual diagram of a carbon dioxide isotope generator and a carbon dioxide isotope trap. FIG. 3 is a diagram showing the 4.5 μm band absorption spectra of ¹⁴ CO ₂ and a competing gas. FIG. 4 is a diagram showing the relationship between CO ² absorption and time from sample introduction for (a) standard glucose aqueous solution, (b) solid standard glucose, and (c) corn stover powder sample. 5A and 5B are diagrams showing the principle of high-speed scanning cavity ring-down absorption spectroscopy using laser light. FIG. 6 is a diagram showing the temperature dependence of the absorption amount Δβ of ¹³ CO ₂ and ¹⁴ CO ₂ in CRDS. FIG. 7 is a conceptual diagram (partially cut away) of a specific embodiment of the optical resonator. 8A and 8B are schematic diagrams of one embodiment of a light generating device, respectively. FIG. 9A is a schematic diagram of a light generating device with double pass. FIG. 10 is a schematic diagram of the vicinity of the optical switch in the light generating device. FIG. 11A and FIG. 11B show the ringdown signal obtained in a single pass and the residual error in fitting using an attenuation function to obtain its attenuation rate. FIGS. 12A and 12B show the ringdown signal obtained by double pass and the residual error in fitting with an attenuation function to obtain its attenuation rate. FIG. 13 shows the sum of squares of residual errors in fitting for each ringdown signal measured for a large number of ringdown signals (variation in the sum of squares of residuals). FIG. 14 is a schematic diagram of a carbon isotope analyzer 1B according to the second embodiment. FIG. 15 is a diagram showing the relationship between absorption wavelength and absorption intensity of analysis samples ¹² C ¹⁶ O ₂ , ¹³ C ¹⁸ O ₂ , ¹³ C ¹⁶ O ₂ , and ¹⁴ C ¹⁶ O ₂ . 16A, 16B, and 16C are schematic diagrams of a second embodiment of the carbon isotope analysis method. teeth, FIG. 17 is a conceptual diagram of a ¹⁴ CO ₂ photosynthetic labeling system for plant samples. FIG. 18 is a diagram showing the acquired spectrum. FIG. 19A and FIG. 19B are diagrams showing respective acquired spectra. FIG. 20A is a diagram in which the vertical axis from FIG. 19A is normalized by the intensity of ¹² CO ₂ , and FIG. 20B is a diagram in which the vertical axis from FIG. 19B is normalized by the intensity at ¹² CO ₂ . FIG. 21 is a diagram plotting d13C measured for each sample based on the results shown in Table 1. FIGS. 22A and 22B are diagrams showing the results of absorption spectra of ¹⁴ C-labeled and non-labeled stem and leaf samples obtained using a ¹⁴ C-CRDS analysis system. FIGS. 23A and 23B are diagrams showing absorption spectra of the root system and foliage of the same individual sampled immediately after the PEG group. FIG. 24A and FIG. 24B are diagrams showing the ¹⁴ C isotope ratio of the stem and leaf parts and the root system with respect to the elapsed time from each labeling. FIGS. 25A and 25B are diagrams showing changes over time of ¹⁴ C in the root system and shoots and leaves as the amount of radioactivity in the sample with respect to the time elapsed from labeling. FIG. 26 is a diagram showing the results of dividing ^{the 14} C isotope ratio of the root system by the ¹⁴ C isotope ratio of the stem and leaves based on the results of FIG. 25. FIG. 27 is a diagram showing the distribution ratio from the root to the shoot for each elapsed time, evaluated from the results of FIG. 25.

The present invention will be described below with reference to embodiments, but the present invention is not limited to the following embodiments. Components having the same or similar functions in the drawings are designated by the same or similar reference numerals, and the description thereof will be omitted. However, the drawings are schematic. Therefore, specific dimensions, etc. should be determined in light of the following description. Furthermore, it goes without saying that the drawings include portions with different dimensional relationships and ratios.

[First aspect of carbon isotope analyzer for plant sample analysis]
The carbon isotope analyzer will be explained using the radioactive isotope ^14C , which is a carbon isotope, as an example of an analysis target. Note that the light having the absorption wavelength of the carbon dioxide isotope ¹⁴ CO ₂ generated from the radioactive isotope ¹⁴ C is light in the 4.5 μm band. Although the details will be described later, it is possible to achieve high sensitivity by combining the absorption line of the substance to be measured, the light generating device, and the optical resonator mode with selectivity.

As used herein, "carbon isotope" means stable carbon isotopes ¹² C, ¹³ C, and radioactive carbon isotope ¹⁴ C, unless otherwise specified. In addition, when the element symbol "C" is simply displayed, it means a mixture of carbon isotopes in their natural abundance ratios.
Stable isotopes of oxygen include ¹⁶ O, ¹⁷ O, and ¹⁸ O, and when the element symbol "O" is displayed, it means a mixture of oxygen isotopes at the naturally occurring ratio.
"Carbon dioxide isotope" means ¹² CO ₂ , ¹³ CO ₂ and ¹⁴ CO ₂ unless otherwise specified. In addition, when simply expressed as "CO ₂ ", it means a carbon dioxide molecule composed of carbon and oxygen isotopes in naturally occurring proportions.

FIG. 1 is a conceptual diagram of a carbon isotope analyzer. FIG. 2 is a conceptual diagram of a carbon dioxide isotope generator and a carbon dioxide isotope trap.
The carbon isotope analyzer 1 includes a carbon dioxide isotope generator 40 , a light generator 20 , a carbon dioxide isotope trap 60 , a spectroscopic device 10 , and an arithmetic device 30 .
The carbon dioxide isotope generating device 40 includes a combustion section that generates a gas containing carbon dioxide isotopes from carbon isotopes, and a carbon dioxide isotope purification section.
The spectroscopic device 10 includes an optical resonator 11 having a pair of mirrors 12a and 12b, and a photodetector 15 that detects the intensity of transmitted light from the optical resonator 11.

<Carbon dioxide isotope generator>
The carbon dioxide isotope generating device 40 is not particularly limited and various devices can be used as long as it is capable of converting carbon isotopes into carbon dioxide isotopes. The carbon dioxide isotope generating device 40 preferably has a function of oxidizing the sample and converting carbon contained in the sample into carbon dioxide. For example, total organic carbon (hereinafter referred to as "TOC") generator, sample gas generator for gas chromatography, sample gas generator for combustion ion chromatography, elemental analyzer (EA), etc. A carbon generator (G) 41 can be used. In consideration of the combustion of the plant sample, it is preferable to use a combustion chamber in which the combustion section is arranged substantially perpendicular to the direction of gravity, as described below.

Figure 3 shows ¹⁴ CO ₂ and competing gas ¹³ CO under the conditions of 273 K, CO ₂ partial pressure 20%, CO partial pressure 1.0 × 10 ^-4 %, and N ₂ O partial pressure 3.0 × 10 ^-8 %. 2 shows the 4.5 μm band absorption spectra of ₂ , CO, and N ₂ O.
By burning the pretreated biological sample, a gas containing the carbon dioxide isotope ¹⁴ CO ₂ (hereinafter also referred to as " ¹⁴ CO ₂ ") can be generated. However, along with the generation of ¹⁴ CO ₂ , contaminant gases such as CO and N ₂ O are also generated. As shown in FIG. 2, these CO and N ₂ O each have an absorption spectrum in the 4.5 μm band, so they compete with the absorption spectrum in the 4.5 μm band that ¹⁴ CO ₂ has. Therefore, in order to improve analytical sensitivity, it is preferable to remove CO and N ₂ O.
Examples of methods for removing CO and N ₂ O include a method of collecting and separating ¹⁴ CO ₂ as described below. Other examples include a method of removing and reducing CO and N ₂ O using an oxidation catalyst or a platinum catalyst, and a combination with the above-mentioned collection and separation method.

(i) Collection and separation of ¹⁴ CO ₂ using thermal desorption column The carbon dioxide isotope generator can include a combustion section and a carbon dioxide isotope purification section. The combustion section can include a combustion tube and a heating section that can heat the combustion tube. Preferably, the combustion tube is made of heat-resistant glass (such as quartz glass) so as to be able to accommodate a sample therein, and a sample inlet is formed in a portion of the combustion tube. In addition to the sample introduction port, the combustion tube may have a carrier gas introduction port so that carrier gas can be introduced into the combustion tube. In addition to providing a sample inlet or the like in a part of the combustion tube, a sample inlet may be formed at one end of the combustion tube using a separate member from the combustion tube, and the sample inlet or carrier gas may be introduced into the sample inlet. It may also be configured to form a mouth.
Examples of the heating section include an electric furnace such as a tubular electric furnace, which can have a combustion tube disposed therein and can heat the combustion tube. An example of a tubular electric furnace is ARF-30M (Asahi Rika Seisakusho).
Further, the combustion tube preferably includes an oxidizing section and/or a reducing section filled with at least one type of catalyst on the downstream side of the carrier gas flow path. The oxidizing section and/or the reducing section may be provided at one end of the combustion tube, or may be provided as separate members. Examples of the catalyst to be filled in the oxidizing section include copper oxide and a mixture of silver and cobalt oxide. In the oxidizing section, it is expected that H ₂ and CO generated by combustion of the sample will be oxidized into H ₂ O and CO ₂ . Examples of the catalyst to be filled in the reduction section include reduced copper and platinum catalysts. In the reduction section, it is expected that nitrogen oxides (NO _x ) containing N ₂ O will be reduced to N ₂ .
As the carbon dioxide isotope purification unit, a thermal desorption column (CO ₂ collection column) such as that used in gas chromatography (GC) can be used to collect ¹⁴ CO ₂ in the gas generated by combustion of a biological sample. . Thereby, the influence of CO and N ₂ O can be reduced or eliminated at the stage of detecting ¹⁴ CO ₂ . Further, by temporarily collecting CO ₂ gas containing ¹⁴ CO ₂ in the GC column, concentration of ¹⁴ CO ₂ can be expected, and therefore an improvement in the partial pressure of ¹⁴ CO ₂ can be expected.
(ii) Trapping of ¹⁴ CO ₂ by ¹⁴ CO ₂ adsorbent and separation of ¹⁴ CO ₂ by re-emission The carbon dioxide isotope generator 40b preferably includes a combustion section and a carbon dioxide isotope purification section. The combustion section can be configured in the same manner as described above.
As the carbon dioxide isotope purification section, ^{a 14} CO ₂ adsorbent such as soda lime or calcium hydroxide can be used. Thereby, the problem of contaminant gas can be solved by isolating ¹⁴ CO ₂ in the form of carbonate. Since it retains ¹⁴ CO ₂ as carbonate, it is also possible to temporarily store the sample. Note that phosphoric acid can be used for re-release.
Contaminant gas can be removed by providing either (i) or (ii) or both configurations.
(iii) Concentration (separation) of ¹⁴ _CO2
¹⁴ CO ₂ generated by combustion of the biological sample diffuses within the pipe. Therefore, detection sensitivity (intensity) may be improved by adsorbing ¹⁴ CO ₂ to an adsorbent and concentrating it. Such concentration can also be expected to separate ¹⁴ CO ₂ from CO and N ₂ O.

FIG. 2 is a conceptual diagram of one embodiment of the carbon dioxide isotope generator 40 and the carbon dioxide isotope trap 60. The carbon dioxide isotope generator 40A includes a combustion furnace 40a arranged substantially perpendicular to the direction of gravity (along the X direction on the screen), a sample boat 40b, a carrier gas supply tank 40c, and a dehumidifier. 40d. The carbon dioxide isotope generator 40A may further include an autosampler.
As the combustion furnace, one having a combustion temperature of about 900° C. can be used. As the sample boat, one made of alumina can be used. As the dehumidifying device 40d, various devices can be used without particular limitation, and for example, a membrane dryer can be used.
Measurements begin by placing samples in the sample boat and inserting them into the combustion furnace. The CO ₂ gas generated from the combustion furnace is carried by a carrier gas (pure oxygen) and introduced into the optical resonator 11 after passing through a dehumidifier.
The EA combustion furnace is arranged vertically, and the sample is combusted by free-falling from the upper sample port to the lower furnace. Therefore, for samples that are likely to dissipate into the surroundings, such as plant samples, it is necessary to insert them into a tin capsule (melting point: approximately 230°C), etc., which has a melting point sufficiently low compared to the furnace temperature (900°C), just like liquid samples. There is. When measuring plants, they are often cut and powdered to average out differences between parts, making it difficult to reliably insert all weighed plant powder into a relatively soft tin capsule. Yes, skilled technique is required. However, by adopting a configuration like the carbon dioxide isotope generating apparatus 40A shown in FIG. 2, the workload can be reduced and the reliability of sample introduction can be improved.

[Confirmation experiment]
A major difference between the carbon dioxide isotope generator 40A and EA is that there is no heated adsorption/desorption column. However, since a CO ₂ trap is introduced into the system as a CO ₂ concentration mechanism, CO ₂ is generated in a time pulse, so there is no major problem in measurement. The following experiment was conducted to demonstrate that there was no problem with measurement.
An apparatus as shown in FIGS. 1 and 2 was prepared, and the wavelength of DFB-QCL was fixed at the ¹³ CO ₂ absorption peak. Then, (a) 2.5 mg/50 μL of a standard glucose aqueous solution, (b) about 2.5 mg of solid standard glucose, and (c) about 2 mg of a corn stover powder sample were introduced. The time profile of _CO2 generation and arrival at the CRDS analysis cell was then evaluated. At this time, a CO ₂ trap system was installed, but it was not immersed in liquid nitrogen. In addition, the timing at which the sample was inserted into the furnace was obtained as a trigger for valve opening and closing in place of the column temperature rise, and was taken as the start time. The obtained results are shown in FIG. In the figure, the horizontal axis represents the time from sample introduction, and the vertical axis represents the amount of ¹³ CO ₂ absorbed in the CRDS analysis cell, which depends on the CO ₂ concentration in the cell.
From this result, it was found that CO ₂ was generated in a time pulse manner in all samples. On the other hand, there was a difference of about 10 seconds between the liquid and the solid in the arrival timing, and a difference in peak shape was observed between glucose and corn stover even though they were the same solid. This is thought to be because the CO ₂ generation time profile in the combustion furnace differs due to differences in sample composition, shape (particle size, etc.), and heat transfer. Such differences in timing and shape are sufficiently small for the CO ₂ trap at the latter stage of the solid sample combustion device, and do not have a large effect on measurements. Moreover, compared to EA, the width of the peak of the time profile measured with the CRDS analysis cell became wider.
This is due to the absence of a heated adsorption/desorption column, and is reasonable as the temporal distribution of combustion in the furnace is observed as is. From the above, it has become clear that CO ₂ in plant samples can be analyzed using the ¹⁴ CCRDS analysis system developed so far by connecting the solid sample combustion device.

<Carbon dioxide isotope trap>
As shown in FIG. 2, the carbon dioxide isotope trap 60 is arranged on the upstream side of the gas supply pipe 69a and the gas supply pipe 69 that sends carbon dioxide isotope from the carbon dioxide isotope generator 40 to the spectrometer 10. A valve 66a, a U-shaped trap pipe 61, a valve 66b disposed on the downstream side of the gas supply pipe 69, and a gas supply pipe 69b branched from the gas supply pipe 69a at the valve 66a and communicating with the optical resonator 11. , a pump P for making the gas supply pipe 69a and the resonator 11 negative pressure, a valve 66c disposed on the gas supply pipe 69b between the resonator 11 and the pump P, and a valve 66c for cooling the trap pipe 61. A Dewar bottle 63 that can be filled with liquid nitrogen 65 is provided.
By controlling the opening and closing of the valves 66a to 66c while operating the pump P, it is possible to control the introduction of the carbon dioxide isotope generated by the carbon dioxide isotope generator into the optical resonator 11.
Carbon dioxide isotope absorber 68 is used to trap ¹⁴ CO ₂ as calcium carbonate so that the carbon dioxide isotope is not exposed to atmospheric release.

<Spectroscopy device>
As shown in FIG. 1, the spectroscopic device 10A includes an optical resonator 11 and a photodetector 15 that detects the intensity of transmitted light from the optical resonator 11. The optical resonator or optical cavity 11 has a cylindrical body in which a carbon dioxide isotope to be analyzed is sealed, and a concave surface is arranged so that one longitudinal end and the other end of the body face each other. It includes a pair of mirrors 12a and 12b with high reflectance, a piezo element 13 that adjusts the interval between the mirrors 12a and 12b arranged at the other end inside the main body, and a cell 16 filled with a gas to be analyzed. Although not shown here, it is preferable to provide a gas injection port for injecting carbon dioxide isotope and an air pressure adjustment port for adjusting the air pressure inside the main body on the side of the main body. Note that the reflectance of the pair of mirrors 12a and 12b is preferably 99% or more, more preferably 99.99% or more.
When a laser beam is incident and confined inside the optical resonator 11, the laser beam repeats multiple reflections on the order of several thousand to ten thousand times while outputting light with an intensity corresponding to the reflectance of the mirror. Therefore, since the effective optical path extends to several tens of kilometers, a large amount of absorption can be obtained even if the amount of gas to be analyzed sealed inside the optical resonator is extremely small.

FIGS. 5A and 5B are diagrams showing the principle of high-speed scanning cavity ring-down absorption spectroscopy (hereinafter also referred to as "CRDS") using laser light.
As shown in FIG. 5A, when the mirror spacing satisfies the resonance condition, a high-intensity signal is transmitted from the optical resonator. On the other hand, if the piezo element 13 is operated to change the mirror spacing to create a non-resonant condition, it becomes impossible to detect a signal due to the light interference effect. That is, by quickly changing the optical resonator length from a resonant condition to a non-resonant condition, an exponential decay signal [Ringdown signal] as shown in FIG. 5A can be observed. Another method for observing the ringdown signal is to quickly shut off the input laser beam using an optical switch.
If the optical resonator is not filled with an absorbing material, the transmitted time-dependent ringdown signal will have a curve as shown by the dotted line in FIG. 5B. On the other hand, when the optical resonator is filled with a light-absorbing material, as shown by the solid line in FIG. 8B, the laser light is absorbed each time it travels back and forth within the optical resonator, so the decay time of the light becomes shorter. The decay time of this light depends on the concentration of the absorbing substance in the optical resonator and the wavelength of the incident laser beam, so the absolute concentration of the absorbing substance can be calculated by applying Beer-Lambert's law II. . Further, by measuring the amount of change in the attenuation rate (ringdown rate), which is proportional to the absorption substance concentration in the optical resonator, the absorption substance concentration in the optical resonator can be measured.

After the transmitted light leaking from the optical resonator is detected by a photodetector and the ¹⁴ CO ₂ concentration is calculated using an arithmetic device, ^{the 14} C concentration can be calculated from the ¹⁴ CO ₂ concentration.

It is preferable that the distance between the mirrors 12a and 12b of the optical resonator 11, the radius of curvature of the mirrors 12a and 12b, the longitudinal length and width of the main body, etc., be changed depending on the absorption wavelength of the carbon dioxide isotope to be analyzed. The assumed resonator length is 1 mm to 10 m.
In the case of the carbon dioxide isotope ^14CO2 , a long cavity length is effective in securing the optical path length, but as the cavity length increases, the volume of the gas cell increases and the amount of sample required increases, so the _resonance The length is preferably between 10cm and 60cm. Further, it is preferable that the radius of curvature of the mirrors 12a and 12b is equal to or longer than the resonator length.
Note that by driving the piezo element 13, the mirror spacing can be adjusted, for example, on the order of several micrometers to several tens of micrometers. Fine adjustments can also be made using the piezo element 13 in order to create optimal resonance conditions.
Although a pair of concave mirrors has been illustrated and explained as the pair of mirrors 12a and 12b, other combinations of concave mirrors and plane mirrors, or combinations of plane mirrors may also be used as long as a sufficient optical path can be obtained. It doesn't matter if there is.
Sapphire glass, CaF ₂ , and ZnSe can be used as materials for forming the mirrors 12a and 12b.
The cell 16 filled with the gas to be analyzed preferably has a smaller volume. This is because the optical resonance effect can be effectively obtained even with a small number of samples to be analyzed. An example of the capacity of the cell 16 is 8 mL to 1000 mL. The cell capacity can be appropriately selected depending on the amount of ¹⁴ C source that can be subjected to measurement. For ¹⁴ C sources that can be obtained in large quantities such as urine, a cell of 80 mL to 120 mL is suitable; For ¹⁴ C sources such as lachrymal fluid, which are available in limited amounts, a cell of 8 mL to 12 mL is suitable.

Evaluation of stability conditions of optical resonator In order to evaluate the ¹⁴ CO ₂ absorption amount and detection limit in CRDS, calculations were performed based on spectral data. Spectroscopic data regarding ¹² CO ₂ , ¹³ CO _2, etc. were obtained using the Atmospheric Absorption Line Database (HITRAN), and regarding ¹⁴ CO ₂ , literature values ("S. Dobos et al., Z. Naturforsch, 44a, 633-639 (1989 )")It was used.
Here, the amount of change Δβ (= β - β ₀ , β: attenuation rate with sample, β ₀ : attenuation rate without sample) in the ringdown rate (rate of exponential decay) due to absorption of ¹⁴ CO ₂ is as follows: It can be expressed as follows using the optical absorption cross section σ ₁₄ of ¹⁴ CO ₂ , the molecular number density N, and the speed of light c.
Δβ=σ ₁₄ (λ,T,P)N(T,P,X ₁₄ )c
(In the formula, σ ₁₄ and N are functions of laser light wavelength λ, temperature T, pressure P, and X ₁₄ = ¹⁴ C/ ^Total C ratio.)
FIG. 6 is a diagram showing the temperature dependence of Δβ due to absorption of ¹³ CO ₂ and ¹⁴ CO ₂ determined by calculation. From Figure 9, when ¹⁴ C/ ^Total C is 10 ^-10 , 10 ^-11 , and 10 ^-12 , the absorption by ¹³ CO ₂ at room temperature 300K exceeds or is about the same as the amount of ¹⁴ CO ₂ absorbed, so cooling is not necessary. I knew I needed to do it.
On the other hand, it can be seen that if the dispersion of the ring-down rate, which is a noise component derived from the optical resonator, can be reduced to Δβ ₀ to 10 ¹ s ⁻¹ , it is possible to measure ^{a 14} C/ ^Total C ratio of 10 ⁻¹¹ . This revealed that cooling to about -40 degrees Celsius was necessary during analysis.
For example, if ¹⁴ C/ ^Total C is set to 10 ^-11 as the lower limit of quantification, it is suggested that an increase in the partial pressure of CO ₂ gas (for example, 20%) due to concentration of CO ₂ gas and the above-mentioned temperature conditions are necessary. .
Note that the cooling device and cooling temperature will be described in more detail in the section of the second embodiment of the carbon isotope analyzer described later.

Although the optical resonator 11 has been described, a conceptual diagram (partially cutaway diagram) of a specific aspect of the optical resonator is shown in FIG. As shown in FIG. 7, the optical resonator 51 includes a cylindrical heat-insulating chamber 58 serving as a vacuum device, a measurement gas cell 56 disposed within the heat-insulation chamber 58, and a gas cell 56 disposed at both ends of the measurement gas cell 56. A pair of high reflectivity mirrors 52, a mirror drive mechanism 55 disposed at one end of the measurement gas cell 56, a ring piezo actuator 53 disposed at the other end of the measurement gas cell 56, and the measurement gas cell 56 are cooled. and a water-cooled heat sink 54 having a cooling pipe 54a connected to a circulating cooler (not shown).

<Arithmetic device>
The arithmetic device 30 is not particularly limited, and may be of various types as long as it can measure the absorbing substance concentration in the optical resonator from the above-mentioned decay time and ring-down rate, and measure the carbon isotope concentration from the absorbing substance concentration. A device can be used.
The arithmetic control section 31 may be constructed of arithmetic means used in ordinary computer systems such as a CPU. Examples of the input device 32 include a keyboard and a pointing device such as a mouse. Examples of the display device 33 include an image display device such as a liquid crystal display and a monitor. Examples of the output device 34 include a printer and the like. As the storage device 35, storage devices such as ROM, RAM, and magnetic disk can be used.

<Light generator>
Although various light sources can be used as the light source 23 without particular limitations, it is preferable to use an ultrashort pulse wave generator. When an ultrashort pulse wave generator is used as the light source 23, the photon density per pulse is high, so nonlinear optical effects easily occur, and light _in the 4.5 μm band, which is the absorption wavelength of the radioactive carbon dioxide isotope ^14CO2 , is generated. can be easily generated. In addition, since a comb-shaped bundle of light (optical frequency comb, hereinafter also referred to as "optical comb") in which the wavelength width of each wavelength is equal is obtained, fluctuations in the oscillation wavelength can be made negligibly small. Note that when a continuous wave generator is used as a light source, the oscillation wavelength varies, so it is necessary to measure the variation in the oscillation wavelength using an optical comb or the like.
As the light source 23, for example, a solid laser, a semiconductor laser, or a fiber laser that outputs short pulses by mode locking can be used. Among these, it is preferable to use a fiber laser. This is because fiber lasers are compact, environmentally stable, and practical light sources.
As the fiber laser, an erbium (Er)-based (1.55 μm band) or ytterbium (Yb)-based (1.04 μm band) fiber laser can be used. From an economic point of view, it is preferable to use a commonly used Er-based fiber laser, and from the perspective of increasing light intensity, it is preferable to use a Yb-based fiber laser.

FIG. 8A shows one embodiment of the light generating device (difference frequency mixing). The light generating device 20A includes one light source 23, a first optical fiber 21 that transmits light from the light source 23, and a first optical fiber that branches from a branch point of the first optical fiber and joins at a downstream junction of the first optical fiber 21. A second optical fiber 22 that transmits light with a longer wavelength than that of the optical fiber 21, and a nonlinear optical crystal 24 that allows a plurality of lights with different frequencies to pass through and generates light with an absorption wavelength of carbon dioxide isotope from the difference in frequency. Be prepared.
The plurality of optical fibers 21 and 22 include a first optical fiber 21 that transmits light from a light source, and a second optical fiber 22 for wavelength conversion that branches from the first optical fiber 21 and joins on the downstream side of the first optical fiber 21. Can be used. As the first optical fiber 21, one that is connected from the light source to the optical resonator can be used. Furthermore, a plurality of optical components and a plurality of types of optical fibers can be arranged on each optical fiber path.
As the first optical fiber 21, it is preferable to use an optical fiber that can transmit the generated high-intensity ultra-short pulse light without deteriorating its characteristics. Specifically, it can include a dispersion compensating fiber (DCF), a double clad fiber, and the like. Preferably, fibers made of fused silica are used as the material.
As the second optical fiber 22, it is preferable to use an optical fiber that can efficiently generate ultrashort pulse light on the desired long wavelength side and transmit the generated high-intensity ultrashort pulse light without deteriorating its characteristics. Specifically, it can include a polarization maintaining fiber, a single mode fiber, a photonic crystal fiber, a photonic bandgap fiber, and the like. It is preferable to use an optical fiber having a length of several meters to several hundred meters, depending on the amount of wavelength shift. Preferably, fibers made of fused silica are used as the material.

The nonlinear optical crystal 24 is appropriately selected depending on the incident light and the emitted light, but in the case of this embodiment, it generates light with a wavelength around 4.5 μm band from each incident light. From this point of view, for example, a PPMgSLT (periodically poled MgO-doped Stoichiometric Lithium Tantalate (LiTaO ₃ )) crystal, a PPLN (periodically poled Lithium Niobate) crystal, or a GaSe (Gallium selenide) crystal can be used. Furthermore, since one fiber laser light source is used, fluctuations in optical frequency can be canceled in difference frequency mixing, as described later.
The nonlinear optical crystal 24 preferably has a length in the irradiation direction (longitudinal direction) of longer than 11 mm, more preferably 32 mm to 44 mm. This is because a high-output optical comb can be obtained.

According to Difference Frequency Generation (hereinafter also referred to as "DFG"), multiple lights with different wavelengths (frequencies) transmitted by the first and second optical fibers 21 and 22 are passed through a nonlinear optical crystal to achieve this effect. From the difference in frequency, light corresponding to the difference frequency can be obtained. In other words, in the case of this embodiment, one light source 23 generates two lights with wavelengths λ ₁ and λ ₂ , and by passing the two lights through a nonlinear optical crystal, carbon dioxide isotopes are determined from the difference in frequency. It can generate light at wavelengths absorbed by the body. The conversion efficiency of a DFG using a nonlinear optical crystal depends on the photon density of the original light source having multiple wavelengths (λ ₁ , λ ₂ , ... λ _x ). Therefore, light of a difference frequency can be generated from one pulse laser light source by the DFG.
The light in the 4.5 μm band obtained in this way is an optical comb (frequency f = f _ceo + N f _r , N: _mode number). In order to perform CRDS using an optical comb, it is necessary to introduce light in the absorption band of the analysis target into an optical resonator containing the analysis target. Note that in the generated optical comb, f _ceo is canceled in the difference frequency mixing process, and f _ceo becomes 0.

In the case of the carbon isotope analysis device devised by I. Galli et al. Irradiation light with the absorption wavelength of carbon dioxide isotope was generated from the difference in light frequencies. Both are continuous wave lasers, and the intensity of ECDL is low, so in order to obtain a DFG with sufficient intensity, a nonlinear optical crystal used in the DFG is installed in an optical resonator, and the light from both is input into it. , it was necessary to increase the photon density. Furthermore, in order to increase the intensity of ECDL, it was also necessary to excite the Ti:Sapphire crystal with a double wave of another Nd:YAG laser to amplify the ECDL light. The resonator that performs these functions must be controlled, making the device large-scale and complicated to operate. On the other hand, since the light generating device according to the embodiment of the present invention is composed of one fiber laser light source, several meters of optical fiber, and a nonlinear optical crystal, it is compact, easy to transport, and easy to operate. . Furthermore, since a plurality of lights are generated from one light source, the fluctuation width and fluctuation timing of each light are the same. Therefore, fluctuations in optical frequency can be easily canceled by performing difference frequency mixing without using a control device.
Regarding the optical path between the confluence of the first optical fiber and the second optical fiber and the optical resonator, the mode of transmitting the laser beam in the air, and the optical system that condenses and/or expands the laser beam with a lens as necessary. An optical transmission device including the following may be constructed.

In this analysis, it is sufficient to obtain an optical comb in a range that covers the wavelength range used in the analysis of ¹⁴ C. Therefore, the inventors believe that it is better to narrow the oscillation spectrum of the optical comb light source to achieve higher output. We focused on the fact that light can be obtained. When the oscillation spectrum is narrow, amplification using amplifiers with different bands or a long nonlinear optical crystal can be used. Therefore, as a result of studies, the present inventors found that in the generation of an optical comb using the difference frequency mixing method, (a) one light source generates multiple lights with different frequencies, and (b) the resulting multiple lights (c) By passing multiple lights through a nonlinear optical crystal that is longer than a conventional nonlinear optical crystal, the absorption wavelength of the carbon dioxide isotope is determined from the difference in frequency. The idea was to generate high-power irradiation light with . The present invention was completed based on the above findings. In addition, in the conventional difference frequency mixing method, there has been no report that the intensity of light can be amplified using multiple amplifiers with different bands or that high-output irradiation light can be obtained using a long crystal.

When the absorption line intensity of a light-absorbing substance is large and the intensity of the irradiated light is also high, the lower level corresponding to the light absorption decreases significantly, and the effective amount of light absorption appears to be saturated. (This is called saturated absorption). According to the SCAR theory (Saturated Absorption CRDS), when a sample such as ¹⁴ CO ₂ in an optical resonator is irradiated with light in the 4.5 μm band with a large absorption line intensity, the initial attenuation signal (ringdown signal) obtained is The saturation effect is large because the light intensity accumulated in the optical resonator is high, and thereafter, as the attenuation progresses, the light intensity accumulated in the optical resonator gradually decreases, so the saturation effect becomes smaller. Therefore, a decay signal in which such a saturation effect is observed is no longer a simple exponential decay. Based on this theory, by fitting the attenuation signal obtained by SCAR, the attenuation rate due to the sample and the attenuation rate of the background can be evaluated independently. Since the attenuation rate due to the sample can be determined without any interference, and the saturation effect of ¹⁴ CO ₂ is greater than that of the contaminant gas, light absorption by ¹⁴ CO ₂ can be measured more selectively. Therefore, it is expected that the sensitivity of analysis will be improved by using irradiation light with higher light intensity. Since the light generating device of the present invention can generate irradiation light with high optical intensity, it is expected that the analytical sensitivity will be improved when used for carbon isotope analysis.

FIG. 8B shows one embodiment (cat eye) of the light generating device. FIG. 2 is a conceptual diagram of a carbon dioxide isotope generator and a carbon dioxide isotope trap. The light generating device 20B includes one light source 23, a first optical fiber 21 that transmits light from the light source 23, and a first optical fiber that branches from a branch point of the first optical fiber and joins at a downstream junction of the first optical fiber 21. a second optical fiber 22 that transmits light with a longer wavelength than that of the optical fiber 21; a nonlinear optical crystal 24 that passes a plurality of lights with different frequencies to generate light with a wavelength absorbed by carbon dioxide isotope from the difference in frequency; It includes an optical switch 25 that controls on/off of the light from the light source 23, and mirrors 26a and 26b that reflect the light from the optical switch 25 and send the light back to the optical switch 25.

<Light generator with double pass>
FIG. 9A is a schematic diagram of a light generating device. The present inventors conducted further studies to further improve the analysis accuracy of the carbon isotope analyzer, and found that the attenuation rate was lower than expected due to the performance (on-off ratio) of the optical switch. It was discovered that an error (residual error in fitting using the attenuation function to determine the attenuation rate of the ringdown signal) was generated in the method. However, no simple and effective mechanism or method for on/off control has been found. Therefore, there has been a need to improve the performance (on-off ratio) of optical switches to eliminate residual errors in ring-down signal fitting and improve analysis accuracy.
According to the present invention, there are provided a light generating device with little residual error in ringdown signal fitting, and a radiocarbon isotope analysis device and a radiocarbon isotope analysis method using the same.
The light generating device 20C includes a light source 23, an optical switch 25 that controls on/off of the light from the light source 23, and mirrors 26a and 26b that reflect the light from the optical switch 25 and send the light back to the optical switch 25. Although there is no particular restriction on the optical path 21, for example, an optical fiber can be arranged.
The light generator 20C further includes mirrors 26c, 26d, and 26e that introduce the light from the optical switch 25 into the optical spectrometer 10A.
As the light source 23, various sources can be used without particular limitation. Details will be described later.
As the optical switch 25, various types can be used without particular limitation, but it is preferable to use an acousto-optic modulator (hereinafter also referred to as "AOM") that includes an optical crystal 25a and a piezoelectric element 25b. .

FIG. 10 is a schematic diagram of the vicinity of the optical switch in the light generating device. As shown in path 1 in FIG. 10, when the piezoelectric element 25b of the AOM is activated, acoustic waves propagate within the optical crystal 25a. This creates a periodic refractive index distribution within the optical crystal, and by diffracting the incident light, it is possible to control on/off of the light from the light source 23. However, even if the light emission is controlled to be turned off, there is a problem in that a small amount of uncontrolled light leaks out and causes an error in the ring-down signal. Therefore, in order to solve the above problem, the present inventors completed a light generation device equipped with a double path in which mirrors 26a and 26b are arranged.

Next, the operation and effects of the light generating device will be explained. (a) As shown in path 1 (P1) in FIG. 10, the light from the light source 23 is sent to the optical switch 25 and controlled on/off using the piezoelectric element 25b. After that, (b) the light leaking from the optical switch 25 is reflected using mirrors 26a and 26b. Furthermore, (c) as shown in path 2 (P2) in FIG. 10, the light sent back to the optical switch 25 is controlled on and off again using the piezoelectric element 25b. In this way, since the light generating device performs on/off control of light with double passes (P1, P2), it is possible to obtain a much higher on/off ratio compared to a single pass, and the light leakage from the optical switch 25 is reduced. Can effectively prevent leakage.

However, since high-speed on/off control is essential for obtaining a ring-down signal, if a double pass is used and light is passed through an arbitrary position, a delay in switching time will occur. Therefore, by transmitting light to positions that are the same distance from the surface of the optical crystal 25a to which the piezoelectric element 25b is attached (P1, P2), it is possible to achieve both a high on-off ratio and high-speed on/off control.

In order to confirm the effects of the light generator provided with double-pass, an experiment was conducted to compare ring-down signals obtained with single-pass and ring-down signals obtained with double-pass under the following conditions. A continuous laser beam with a wavelength of 4.5 μm was controlled on and off by a light generator, and was introduced into an optical resonator not filled with gas to obtain a ring-down signal. The obtained results are shown in FIGS. 11 and 12.
FIG. 11 shows a ringdown signal obtained by a single pass, and FIG. 12 shows a ringdown signal obtained by a double pass. In the case of the single pass shown in FIG. 11, the oscillation width of the residual error was large up to the first 10 μs of the ring-down signal. On the other hand, in the case of the double pass shown in FIG. 12, the problem of the vibration width of the initial residual was resolved, and the fluctuation width of the vibration width throughout the ring-down signal was narrower than in the case of FIG. 11.
FIG. 13 shows the sum of squares of residual errors in fitting to each ringdown signal measured for a large number of ringdown signals, that is, the variation in residual errors. Compared to the single-pass residual shown in the upper part of FIG. 13, the double-pass residual shown in the lower part was smaller.

Although the carbon isotope analyzer according to the first aspect has been described above, the carbon isotope analyzer is not limited to the above-described embodiment and can be modified in various ways. Another aspect of the carbon isotope analyzer will be described below, focusing on changes from the first aspect.

[Second embodiment of carbon isotope analyzer for plant sample analysis]
Conventionally, quantum cascade lasers (hereinafter also referred to as "QCL") have fluctuations in their oscillation wavelengths, and the absorption wavelengths of ¹⁴ C and ¹³ C are adjacent, so carbon isotope analysis such as that used for ¹⁴ C analysis has been difficult. It was thought that it would be difficult to use it as a light source for devices. Therefore, the present inventors independently developed an optical comb light source that generates an optical comb from a single light source, thereby completing a compact and easy-to-use carbon isotope analyzer (see Patent Document 2).
In order to further improve the analytical accuracy of the carbon isotope analyzer, the present inventors have completed a light generating device that generates high-output (high-intensity) light with a narrow line width, as described above. As a result of considering further applications of the light generating device, the present inventors have determined that fluctuations in the oscillation wavelength of the light emitted from the QCL can be measured using a beat signal measuring device that uses the narrow linewidth light generated from the above-mentioned light generating device as a frequency reference. I thought of making a correction. As a result of conducting research based on this idea, we completed a compact, easy-to-use, and highly reliable light generator that uses a light source other than an optical comb as the main light source, and a carbon isotope analyzer using it.

FIG. 14 is a diagram showing an outline of a carbon isotope analyzer 1B according to the second embodiment. The carbon isotope analyzer 1B in FIG. 14 is the same as the light generator 20A in FIG. 1, except that the light generator 20 and spectrometer 10 in FIG. 1 are replaced with the light generator 20B and spectrometer 10B in FIG. It has the following configuration.
The light generating device 20D includes a light source 23, an optical switch 25 that controls on/off of the light from the light source 23, and mirrors 26a and 26b that reflect the light from the optical switch 25 and send the light back to the optical switch 25. Although there is no particular restriction on the optical path 21, for example, an optical fiber can be arranged.
The light generator 20 further includes mirrors 26c, 26d, and 26e that introduce the light from the optical switch 25 into the optical spectrometer 10A.
As the light source 23, various sources can be used without particular limitation. Details will be described later.
As the optical switch 25, various types can be used without particular limitation, but it is preferable to use an acousto-optic modulator (hereinafter also referred to as "AOM") that includes an optical crystal 25a and a piezoelectric element 25b. .
The light generating device 20D includes a main light source 23B and a beat signal measuring device 28.
As the main light source 23D, a general-purpose light source such as a QCL can be used.
The beat signal measuring device 28 includes an optical comb source 28a that generates an optical comb consisting of a bundle of light with a narrow linewidth in which the frequency range of one light is 4500 nm to 4800 nm, and the light from the main light source 23 and the optical comb source 28a. and a photodetector 28b that measures a beat signal caused by a frequency difference between the lights. The light source in the first embodiment described above can be used as the optical comb source 28a.
A part of the light from the main light source 23 is sent to the photodetector 28b through a branching means 29a arranged on the optical fiber 21 and a branching means 29b arranged on the optical axis of the light from the optical comb source 28a. By sending in the light, a beat signal can be generated due to the frequency difference between the light from the main light source 23 and the light from the optical comb source 28a.
In the carbon isotope analyzer 1B equipped with the light generator 20B, the main light source is not limited to an optical comb, and a general-purpose light source such as a QCL can be used, allowing freedom in design and maintenance of the carbon isotope analyzer 1B. The degree becomes higher.
As the light generating device 20B, various devices can be used without particular limitation as long as they can generate light having an absorption wavelength of carbon dioxide isotope. Here, a light generation device that easily generates light in the 4.5 μm band, which is the absorption wavelength of the radioactive carbon dioxide isotope ¹⁴ CO ₂ and is compact in size, will be described as an example.

<Cooling and dehumidification equipment>
As shown in FIG. 14, the spectroscopic device 1a may further include a Peltier element 19 that cools the optical resonator 11, and a vacuum device 18 that houses the optical resonator 11. Since the optical absorption of ¹⁴ CO ₂ has temperature dependence, by lowering the set temperature in the optical resonator 11 using the Peltier element 19, the absorption line of ¹⁴ CO ₂ and the absorption lines of ¹³ CO ₂ and ¹² CO ₂ can be changed. This is because it becomes easier to distinguish between the two, and the absorption intensity of ¹⁴ CO ₂ becomes stronger. Furthermore, the analytical accuracy is improved by arranging the optical resonator 11 within the vacuum device 18 to prevent the optical resonator 11 from being exposed to the outside air and to reduce the influence of external temperature.
As a cooling device for cooling the optical resonator 11, in addition to the Peltier element 19, for example, a liquid nitrogen tank, a dry ice tank, etc. can be used. From the viewpoint of miniaturizing the spectroscopic device 10, it is preferable to use the Peltier element 19, and from the viewpoint of reducing the manufacturing cost of the device, it is preferable to use a liquid nitrogen water tank or a dry ice tank.
The vacuum device 18 is not particularly limited as long as it can house the optical resonator 11, irradiate the light from the light generator 20 into the optical resonator 11, and transmit transmitted light to the photodetector. Various vacuum devices can be used.
A dehumidifier may also be provided. At that time, dehumidification may be performed using a cooling means such as a Peltier element, but it may also be dehumidified by a membrane separation method using a water vapor removal polymer membrane such as a fluorine-based ion exchange resin membrane.

When the above-described carbon isotope analyzer 1 is used for microdosing, the detection sensitivity for the radioactive carbon isotope ^14C is assumed to be about "0.1 dpm/ml". In order to achieve this detection sensitivity of "0.1 dpm/ml", it is insufficient to simply use a "narrow band laser" as a light source, and stability of the wavelength (frequency) of the light source is required. However, in plant sample analysis, the amount of radioactivity to be administered is not as limited as in humans, so the lower detection limit of radioactivity and the abundance sensitivity can be solved by administering the amount so that it exceeds the lower limit of the analyzer. Therefore, the lower limit of the abundance sensitivity (analyzable isotope ratio) is about 10 ^-12 (natural isotope ratio).
Although plant sample analysis does not require higher sensitivity than biological samples, it is preferable that the absorption line does not deviate from the wavelength of the absorption line and that the line width is narrow. In this regard, the carbon isotope analyzer 1 can solve this problem by using a stable light source using "optical frequency comb light" for CRDS. The carbon isotope analyzer 1 has the advantageous effect of being able to measure even samples containing low concentrations of radioactive carbon isotopes.
In addition, the previous literature (Kazuro Hiromoto et al., "Design study of 14C continuous monitoring based on cavity ring-down spectroscopy", Atomic Energy Society of Japan spring annual meeting proceedings, March 19, 2010, p. 432) contains information related to nuclear power generation. In connection with the concentration monitoring of spent fuel, it is disclosed that ^{the 14} C concentration in carbon dioxide is measured by CRDS. However, although the signal processing method using fast Fourier transform (FFT) described in the prior literature speeds up data processing, the fluctuation of the baseline becomes large, so detection sensitivity of "0.1 dpm/ml" is not achieved. It is difficult to do so.

Figure 15 (quoted from Applied Physics Vol. 24, pp. 381-386, 1981) shows the absorption wavelengths of analysis samples ¹² C ¹⁶ O ₂ , ¹³ C ¹⁸ O ₂ , ¹³ C ¹⁶ O ₂ , and ¹⁴ C ¹⁶ O ₂ The relationship between absorption intensity is shown. As shown in FIG. 15, carbon dioxide containing each carbon isotope has a unique absorption line. In actual absorption, each absorption line has a finite width due to the spread caused by the pressure and temperature of the sample. For this reason, it is preferable that the pressure of the sample is below atmospheric pressure and the temperature is below 273K (0°C).

As mentioned above, since the absorption intensity of ¹⁴ CO ₂ is temperature dependent, it is preferable to set the temperature inside the optical resonator 11 as low as possible. Specifically, the temperature set inside the optical resonator 11 is preferably 273 K (0° C.) or lower. The lower limit is not particularly limited, but from the standpoint of cooling effect and economy, it is preferable to cool to about 173K to 253K (-100°C to -20°C), particularly about 233K (-40°C).
The spectroscopic device may further include vibration absorption means. This is because it is possible to prevent the mirror interval from shifting due to external vibrations of the spectrometer, thereby increasing measurement accuracy. As the vibration absorbing means, for example, a shock absorber (polymer gel) or a seismic isolation device can be used. As the seismic isolation device, it is possible to use a device that can apply vibrations in the opposite phase to external vibrations to the spectrometer.

[First aspect of carbon isotope analysis method]
This will be explained using the radioactive isotope ^14C as an example of an analysis target.

(a) First, a carbon isotope analyzer 1 as shown in FIGS. 1 and 8A is prepared. In addition, a plant sample containing ¹⁴ C is prepared as a radioactive isotope ¹⁴ C source. After the biological activity of the plant sample has ceased, each part of the plant sample is cut (sampled). Preferably, the plant sample is then dried. Alternatively, the plant sample may be cut into small pieces.

(b) Heating and burning the plant sample to produce a gas containing the carbon dioxide isotope ¹⁴ CO ₂ from the radioactive isotope ¹⁴ C source. In this case, for example, a plant sample can be used that has been photosynthesized in an environment containing ¹⁴ CO ₂ gas using the system described below. N ₂ O and CO are removed from the obtained gas.

(c) It is preferable to remove moisture from the obtained ¹⁴ CO ₂ . For example, in the carbon dioxide isotope generating device 40, it is preferable to remove moisture by passing ¹⁴ CO ₂ over a desiccant such as calcium carbonate or by cooling ¹⁴ CO ₂ to condense moisture. This is because a reduction in mirror reflectance due to icing and frosting of the optical resonator 11 caused by moisture contained in ¹⁴ CO ₂ lowers detection sensitivity, and thus removing moisture improves analysis accuracy. Note _that in consideration of the spectroscopy process, it is preferable to cool ^{the 14} CO ₂ before introducing it into the spectroscopic device 10. This is because when ¹⁴ CO ₂ at room temperature is introduced, the temperature of the resonator changes significantly and the analysis accuracy decreases.

(d) ¹⁴ CO ₂ is filled into an optical resonator 11 having a pair of mirrors 12a and 12b as shown in FIG. Then, it is preferable to cool ^{the 14} CO ₂ to 273 K (0° C.) or lower. This is because the absorption intensity of the irradiated light increases. Further, it is preferable to maintain the optical resonator 11 in a vacuum atmosphere. This is because measurement accuracy is increased by reducing the influence of external temperature.

(e) As shown in FIG. 8A, the first light obtained from the light source 23 is transmitted to the first optical fiber 21. In addition, the first light is transmitted to a second optical fiber 22 that branches from the first optical fiber 21 and joins at a confluence point on the downstream side of the first optical fiber 21, and the second optical fiber 22 transmits a second light having a longer wavelength than the first light. to occur. Note that the intensities of the obtained first light and second light may be amplified using amplifiers (not shown) having different bands.
Then, the first optical fiber 21 on the shorter wavelength side generates light in the 1.3 μm to 1.7 μm band, and the second optical fiber 22 on the longer wavelength side generates light in the 1.8 μm to 2.4 μm band. Next, the second light is combined on the downstream side of the first optical fiber 21, the first light and the second light are passed through the nonlinear optical crystal 24, and from the difference in frequency, the absorption wavelength of carbon dioxide isotope ¹⁴ CO ₂ is 4. As the light in the 5 μm band, an optical comb having a mid-infrared optical frequency in the wavelength band of 4.5 μm to 4.8 μm is generated as the irradiation light. In this case, by using a long-axis crystal with a longitudinal length longer than 11 mm as the nonlinear optical crystal 24, high-intensity light can be generated.

(f) Irradiate the carbon dioxide isotope ¹⁴ CO ₂ with irradiation light and cause it to resonate. At this time, in order to improve measurement accuracy, it is preferable to absorb vibrations from the outside of the optical resonator 11 so that no deviation occurs in the distance between the mirrors 12a and 12b. Further, it is preferable to irradiate the irradiated light while bringing the other downstream end of the first optical fiber 21 into contact with the mirror 12a so that the irradiated light does not come into contact with the air. Then, the intensity of the transmitted light from the optical resonator 11 is measured. Note that the transmitted light may be split into spectra, and the intensity of each of the split transmitted lights may be measured.

(g) Calculate the carbon isotope ^14C concentration from the intensity of transmitted light.

Although the carbon isotope analysis method according to the first aspect has been described above, the carbon isotope analysis method is not limited to the above-described embodiments, and various changes can be made. Another aspect of the carbon isotope analysis method will be described below, focusing on changes from the first aspect.

[Second embodiment of carbon isotope analysis method]
The second aspect of the carbon isotope analysis method is one in which the above-mentioned step (e) is replaced with the following step.
(a) The carbon isotope analysis method generates an optical comb consisting of a bundle of light with a narrow linewidth in the frequency range of 4500 nm to 4800 nm.
(b) Next, as shown in FIG. 16A, the spectrum of one of the optical combs is displayed at the center of the absorption wavelength region of the object to be examined in the optical spectrum diagram of intensity versus frequency.
(c) Transmit the light from the optical comb to the optical fiber for beat signal measurement.
(d) Light from a light source is irradiated onto the object to be inspected, and the amount of light absorption is measured using an optical resonator (CRDS).
(e) Part of the light from the light source is branched to an optical fiber for measuring beat signals, and a beat signal is generated by the frequency difference between the light from the light source and the light from the optical comb source. At this time, the beat signal may be generated while scanning a wide range of frequencies (1), (2), etc. as shown by the arrows in FIG. 16B. Alternatively, a beat signal may be generated in a desired frequency range as shown in FIG. 16C.
(f) Record the wavelength of the light irradiated onto the test object obtained from the beat signal obtained in step (e) together with the amount of light absorption obtained in step (d). Based on these records, the exact amount of light absorption of the object to be tested is measured.
Although the present invention does not purposely perform phase locking using an optical comb, accurate measurement can be achieved with a simple measurement system.

[14CO2 labeling system for plant samples]
The 14CO2 photosynthesis labeling system for plant samples will be described below. In order to conduct experiments to evaluate the movement of photosynthetic products produced in the above-ground parts of rice to the roots, it is desirable to introduce plant samples into a 14CO2 environment and label them with 14C through photosynthesis. Therefore, previous research (R. Sugita, et al., “Visualization of uptake of mineral elements and
the dynamics of photosynthates in arabidopsis by a newly developed real-time
14C-labeled sodium bicarbonate solution (NaH14CO3, 37 MBq/mL, PerkinElmer) and weak acid (lactic acid, CH3CH). We constructed a labeling system using the chemical reaction of (OH)COOH).The outline is shown in Fig. 17.
FIG. 17 is a conceptual diagram of a 14CO2 photosynthetic labeling system 80 for plant samples. The 14CO2 photosynthetic labeling system 80 for plant samples includes a vial 81 for generating 14CO2 gas, a sample bag 83 for accumulating the generated gas, a desiccator 85 into which the plant sample is introduced, and a circulation pump 86. It further includes a pipe 88a that connects each member in series, and a pipe 88b that connects between the circulation pump 86 and the sample bag 83. A valve 89a is provided between the vial 81 and the sample bag 83, a valve 89b is provided between the sample bag 83 and the desiccator 85, and a valve 89c is provided in the pipe 88b. Gas can be circulated between the sample bag 83 and the desiccator 85 by opening and closing each of the valves 89a to 89c and operating the circulation pump 86. A light source 82 is arranged above the desiccator 85. Desiccator 85 can be made of acrylic.
It is preferable that the volume of the sample bag 83 is sufficiently smaller than the volume of the desiccator 85. Although there is no particular limitation, when the volume of the sample bag 83 is 600 mL, the volume of the desiccator 85 can be 30 L. This is because all the gases in the sample bag 83 can be easily introduced into the desiccator 85.

Next, a method for generating ¹⁴ CO ₂ gas will be explained. When lactic acid is dropped into the ¹⁴ C-labeled sodium bicarbonate solution in the vial 81, ¹⁴ CO ₂ gas is generated by the following chemical reaction.
_NaH14CO3 + _CH3CH (OH) _COOH
→ ¹⁴ CO ₂ + H ₂ O + CH ₃ CH(OH)COONa
At this time, if the amount of lactic acid is sufficiently larger than the amount of sodium hydrogen carbonate, all of the ¹⁴ C in the vial 81 becomes ¹⁴ CO ₂ gas, and the generated ¹⁴ CO ₂ gas is converted into ¹⁴ C-hydrogen carbonate present in the vial 81. It can be easily calculated from the amount of sodium solution.

The operation of the above system will be explained. (a) First, open and close the valves 89a to 89c so that only the vial 81 and sample bag 83 are connected. (b) After accumulating ^{the 14} CO ₂ gas generated from the vial 81 in the sample bag 83, the valve is switched to connect only the sample bag 83 and the desiccator 85, and the desiccator 85 is filled with ¹⁴ CO ₂ gas. (c) Thereafter, the circulation pump 86 is used to circulate the gas within the system. As a result, the plant sample inside the desiccator 85 incorporates ¹⁴ CO ₂ into the body through photosynthesis. (d) It is preferable to promote photosynthesis of the plant sample inside the desiccator 85 using a light source 82 (in this case, an LED light) installed at the top of the desiccator 82.

[Plant sample analysis system]
The present invention also relates to a plant sample analysis system comprising the above-described carbon isotope analyzer for plant sample analysis and a ¹⁴ CO ₂ photosynthesis labeling system (unit) for plant samples. According to such a system, plant sample analysis can be performed efficiently. Although each member is preferably located in the same campus, the present invention is not limited thereto, and each member may be located in a separate campus.

(Other embodiments)
As described above, the present invention has been described by way of embodiments, but the statements and drawings that form part of this disclosure should not be understood as limiting the present invention. Various alternative embodiments, implementations, and operational techniques will be apparent to those skilled in the art from this disclosure.
In the carbon isotope analyzer according to the embodiment, the radioactive isotope ^14C has been mainly described as the carbon isotope to be analyzed. In addition to the radioactive isotope ¹⁴ C, stable isotope elements ¹² C and ¹³ C can be analyzed. As the irradiation light in that case, for example, when ¹² C and ¹³ C analysis is performed as absorption line analysis of ¹² CO ₂ and ¹³ CO ₂ , it is preferable to use light in the 2 μm band or 1.6 μm band.
When performing absorption line analysis of ¹² CO ₂ and ¹³ CO ₂ , it is preferable that the mirror spacing be 10 to 60 cm, and that the radius of curvature of the mirror be equal to or greater than the mirror spacing.
Although ^12C , ^13C , and ^14C each exhibit the same chemical behavior, the natural abundance ratio of the radioactive isotope ^14C is lower than that of the stable isotope elements ^12C and ^13C . By artificially increasing the concentration of ¹⁴ C and performing accurate measurements, it becomes possible to observe various reaction processes.
The light generating device (optical switch) described in the first embodiment can control the on/off of light with high precision, so it can be used for various purposes. For example, measuring devices, medical diagnostic devices, environmental measuring devices (dating devices), etc. that partially include the configuration described in the first embodiment can also be manufactured.
The optical frequency comb described in the first embodiment is a light source in which the longitudinal modes of the laser spectrum are arranged at equal frequency intervals with very high precision, and can be used as a new high-performance light source in the fields of precision spectroscopy and high-precision distance measurement. It is expected. Furthermore, since the absorption spectra of substances are mostly in the mid-infrared region, it is important to develop optical frequency comb light sources in the mid-infrared region. The optical frequency comb can be used for various purposes other than those described in the first and second embodiments.
Thus, it goes without saying that the present invention includes various embodiments not described here. Therefore, the technical scope of the present invention is determined only by the matters specifying the invention in the claims that are reasonable from the above description.

[Measurement of ¹³ C-labeled corn sample]
Basic experiments were conducted using ¹³ C-labeled corn samples. The samples were 13C-labeled corn stover and root system samples for purposes other than this research, and were provided by Dr. Mana Nakata of the Graduate School of Bioagricultural Sciences, Nagoya University. The sample had been finely cut and powdered from the beginning. There are 6 samples in total, 3 samples each with and without ^{the 13} C label. Samples with the 13 C label are Nos. 1 to 3 (1, 2: stem and leaf parts, 3: root system), and samples without the ¹³ C label are Nos. 4 to 3. It was labeled as 6 (4, 5: stem and leaf parts, 6: root system).
Furthermore, an acetanilide (C ₈ H ₉ NO) sample (solid) was measured as a reference sample for natural isotope ratio. The ¹³ C isotope ratio of this sample had been analyzed using an isotope ratio mass spectrometer (IRMS) prior to this study. Approximately 2 mg of each sample was weighed and placed in an alumina sample boat, which was then inserted into a solid sample combustion device. First, in order to confirm that CO ₂ absorption spectra derived from plant samples can be obtained using the ¹⁴ C-CRDS analysis system, unlabeled sample No. 5 (stems and leaves) was introduced, and CO 2 absorption spectra containing ¹² C and ¹³ C were introduced. The absorption spectrum in a relatively wide range was measured by scanning the wave number region of 2209.25 to 2210.0 cm ^-1 where carbon has relatively large absorption. The temperature of the gas cell was kept at 283 K and the pressure during the measurements was 20 mbar. The spectrum obtained for ¹³ C-labeled corn stover is shown in FIG. 18.
Although absorption peaks of N ₂ O, CO, and H ₂ O were confirmed, a clear absorption peak of ¹³ CO ₂ and ¹² CO ₂ was obtained without being buried in them. This indicates that it is possible to measure CO ₂ absorption spectra derived from plant samples. Thereafter, the scan range was set to 2209.75 to 2210.0 cm ^-1 where both ¹³ CO ₂ and ¹² CO ₂ peaks were present.

Next, spectra were obtained for all samples (Nos. 1 to 6) and acetanilide samples, and their isotope ratios were evaluated. The temperature of the gas cell was kept at 283 K and the pressure during the measurements was 20 mbar. Examples of the acquired spectra (No. 1 and No. 5, both stem and leaf parts) are shown in FIGS. 19A and 19B, respectively. Here, in order to clarify the change in peak intensity, the vertical axis in the figure is not a logarithmic scale but a linear scale. 20A and 20B are normalized vertical axes based on the intensity of ¹² CO ₂ from FIGS. 19A and 19B because there are individual differences in the carbon content in the samples due to slight differences in the amount of sample introduced.

表１に示された結果をもとに、試料ごとに測定されたｄ１３Ｃをプロットした結果を図２１に示す。この時、縦軸はアセトアニリドの結果により補正した。^１４Ｃ－ＣＲＤＳ分析システムによる評価は、ＩＲＭＳの評価とよく一致した。^１４Ｃ－ＣＲＤＳ分析システムにより測定可能な波数領域（２２０８ｃｍ^－１～２２１２ｃｍ^－１）に存在する^１３ＣＯ_２・^１２ＣＯ_２の吸収線は^１４ＣＯ_２吸収線と比較して吸収断面積が低く、感度には限りがあるものの、本手法によって植物中安定炭素の同位体比を見積もれることが示された。 This shows that ¹³ C-labeled sample No. 1 clearly has a higher relative intensity of ¹³ CO ₂ absorption than unlabeled sample No. 5. Furthermore, while No. 5 was the natural isotope ratio, No. 1 was about twice as large as the evaluated isotope ratio. All samples were measured three times (n3), and the evaluated isotope ratios are shown in Table 1 together with the evaluation results by IRMS. In the table, d13C is the value of the standard sample (Pee Dee Belemnite ore: PDB) of natural ¹³ C isotope ratio ( ¹³ C/ ¹² C 0.012021%) (J. Meija, et al., “Isotopic compositions of the elements 2013 (IUPAC Technical Report)”, Pure Appl. Chem., 88, 3, 293-306 (2016)), and is expressed by the following formula. Note that the unit is parts per thousand (per mil, ‰).

Based on the results shown in Table 1, d13C measured for each sample was plotted and the results are shown in FIG. At this time, the vertical axis was corrected based on the results for acetanilide. The evaluation by the ¹⁴ C-CRDS analysis system was in good agreement with the IRMS evaluation. ^{The 13} CO ₂ ^.12 CO ₂ absorption line that exists in the wave number region (2208 cm ⁻¹ to 2212 cm ⁻¹ ) that can be measured by the ¹⁴ C-CRDS analysis system has a lower absorption cross section than ^{the 14} CO ₂ absorption line. Although the sensitivity is limited, this method was shown to be able to estimate the isotope ratio of stable carbon in plants.

[ ¹⁴ _CO2 labeling system for plant samples]
In order to confirm ¹⁴ C labeling of plant samples, an experiment was conducted in which rice samples grown under the same conditions were labeled using a ¹⁴ CO ₂ photosynthetic labeling system. 20 μL (=740 kBq) of ¹⁴ C-sodium hydrogen carbonate solution was used to generate ¹⁴ CO ₂ gas, and the rice samples were exposed (labeled) to ¹⁴ CO ₂ gas for 50 minutes. One individual was not labeled for comparison of results. After labeling, the plants were separated into stems, leaves and roots, immersed in liquid nitrogen to stop biological activity, and then dried in a dryer. The sample was cut into pieces, and an absorption spectrum was obtained using a ¹⁴ C-CRDS analysis system.
The results for ¹⁴ C-labeled and unlabeled stem and leaf samples are shown in FIGS. 22A and 22B, respectively. The results showed that a clear peak of ¹⁴ CO ₂ that was not observed in the unlabeled sample was measured in the labeled sample, indicating that ¹⁴ CO ₂ labeling by photosynthesis is possible with this system.

[Demonstration of carbon dynamics evaluation of rice by ^14C tracer analysis]
We confirmed that plant sample analysis is possible using the ¹⁴ C-CRDS analysis system described above, constructed a ¹⁴ C labeling system using photosynthesis, and confirmed its operation. Therefore, an experiment was conducted in which rice samples were allowed to absorb ¹⁴ CO ₂ through photosynthesis, and the dynamics of the movement of photosynthetic products to the roots was evaluated. As rice samples, two groups (20 individuals each, 40 individuals in total) of rice (variety: Nipponbare) that were grown for 10 days under hydroponic conditions in an artificial climate machine were prepared. One was grown normally in an artificial climate machine (Control), and the other was artificially subjected to drought stress by mixing polyethylene glycol (PEG) into the hydroponic solution. PEG can absorb a large amount of water internally and can suppress water absorption into plants. All individuals in the Control group and PEG group were introduced into the desiccator of the labeling system shown in FIG. 17 described above.

Since the number of individuals was very large compared to the above experiment, 1 mL (=5.5 MBq) of ¹⁴ C-sodium hydrogen carbonate solution was used to generate ¹⁴ CO ₂ gas, and the rice sample was converted to ¹⁴ CO ₂ gas. It was exposed for 50 minutes. After that, the samples were taken out from inside the desiccator, grouped into groups of 5 each for Control and PEG, and after a certain period of time (immediately, 30 minutes, 60 minutes, and 120 minutes) were removed from the shoot and root system. After separating them, they were each immersed in liquid nitrogen to stop biological activity. This task required about 5 minutes, and the median value was taken as the time taken to stop biological activity.
Thereafter, it was dried in a dryer, weighed individually, and cut and powdered. This kind of work is called sampling. 1.0 mg was measured from the stems and leaves, which are expected to have a relatively large amount of ¹⁴ C, and 1.5 mg was measured from the roots, and absorption spectra were obtained using a ¹⁴ C-CRDS analysis system. The gas cell was maintained at 283 K, and the pressure during the measurements was approximately 15 mbar in the root system and approximately 50 mbar in the stem and leaves. The high pressure in the stem and leaves is due to the large amount of ¹⁴ C and absorption saturation (P. Cancio, et al., “Saturated-Absorption Cavity Ring-Down (SCAR) for High-Sensitivity and High-Resolution Molecular Spectroscopy in This is because the influence of "The Mid IR", Cavity-Enhanced Spectroscopy and Sensing, pp. 143-162, Springer Berlin Heidelberg. (2014)) was noticeable. When saturation occurs, the absorption peak intensity no longer depends on the amount of ¹⁴ C, and the zenith of the peak becomes lower than it should be. The effect of absorption saturation was reduced by increasing the gas pressure and increasing the pressure spread of the absorption spectrum. As examples of the acquired absorption spectra, the results of the root system and stem and leaves of the same individual sampled immediately after the PEG group are shown in FIGS. 23A and 23B, respectively.

ここで、ＮＡはアボガドロ定数（=６．０２２×１０^２３）、λは^１４Ｃ崩壊定数である。横軸に標識からの経過時間、縦軸に試料中放射能量として、根系、茎葉部中の^１４Ｃ時間変化を図２５Ａ、図２５Ｂに示す。また、根系への分配率Drootは茎葉部と根系の放射能量の和（全放射能量）と根系の放射能量の比であり、以下の式で計算される。

ここでAroot・Ashoot はそれぞれ根系・茎葉部で評価された放射能量である。経過時間ごとの分配率を図２７に示す。結果より、ＰＥＧ（乾燥ストレス有）は全体的に放射能量の値が低くなった。これは乾燥ストレスによって、茎葉部の成長や光合成に関する機能が低下していることを示している。実際、ＰＥＧ群の試料は茎葉部の大きさ・重さが相対的に小さくなっており、図２４Ａ、２４Ｂ，図２５Ａ、２５Ｂに示されるように取り込まれた^１４ＣＯ_２が相対的に少ない。一方で、根系の^１４Ｃ同位体比・放射能量・根への分配率の傾きはＰＥＧがControlより大きくなった。これは乾燥ストレスに適応するために光合成産物がより根に送られていることを示している。一方で、ＰＥＧの結果では根系の^１４Ｃ同位体比・放射能量・根への分配率において、飽和の傾向が見られた。根は茎葉部から光合成産物を受け取るだけではなく、それを呼吸によりＣＯ_２として体外に排出する。乾燥ストレス下において、根の呼吸速度が増加することが知られており(仲田（狩野） 麻奈ら、「イネ根系の土壌乾燥ストレス応答に関与する通気組織形成と根呼吸特性」、第２４３回日本作物学会講演会、ｐ.１９２（２０１７）参照)、茎葉部から根への^１４Ｃ供給速度と、呼吸による^１４Ｃ排出速度が平衡状態に達した可能性が示唆された。ＣＲＤＳは気体を分析する手法であり、今後、本実験と同様の評価に加えて、根系からの呼気中の^１４ＣＯ_２量を評価するといった、これまでにない新たな評価法の開発が期待される。 The stem, leaf and root systems of five individuals in each group were measured, and the ^14C isotope ratio was evaluated. The horizontal axis shows the elapsed time from labeling, and the vertical axis shows the ¹⁴ C isotope ratio, and the results are divided into root system and stem and leaf parts and plotted in FIG. 24A and FIG. 24B, respectively. Furthermore, a diagram showing the ratio of isotope ratios between the root system and the stem and leaves, and similarly plotting the elapsed time from the labeling on the horizontal axis, is shown in FIG. Comparing the isotope ratios evaluated in samples from the shoot and root system, it was found that the isotope ratio in the root system is several orders of magnitude lower, and the difference decreases as time elapses (the ratio between roots and leaves increases). . It is reasonable that ¹⁴ C absorbed by photosynthesis in the stem and leaves is transferred to the root system. Furthermore, ^{the 14} C isotope ratio can be converted into the amount of radioactivity A (unit: Bq) in the sample using the following formula.

Here, NA is Avogadro's constant (=6.022×10 ²³ ), and λ is the ¹⁴ C decay constant. Figures 25A and 25B show changes over time of ¹⁴ C in the root system, shoots, and leaves, with the horizontal axis representing the elapsed time from labeling and the vertical axis representing the amount of radioactivity in the sample. In addition, the distribution rate to the root system, Droot, is the ratio of the sum of the radioactivity amounts in the stem and leaf parts and the root system (total radioactivity amount) to the radioactivity amount in the root system, and is calculated by the following formula.

Here, Aroot and Ashoot are the amounts of radioactivity evaluated in the root system and stem and leaves, respectively. FIG. 27 shows the distribution ratio for each elapsed time. The results showed that PEG (with drying stress) had a lower overall radioactivity value. This indicates that drought stress reduces the growth and photosynthetic functions of the shoots and leaves. In fact, the size and weight of the stems and leaves of the PEG group samples are relatively small, and as shown in FIGS. 24A, 24B, 25A, and 25B, ¹⁴ CO ₂ taken in is relatively small. On the other hand, the slopes of the ^14C isotope ratio in the root system, the amount of radioactivity, and the distribution rate to the roots were larger in PEG than in Control. This indicates that more photosynthetic products are being sent to the roots to adapt to drought stress. On the other hand, the PEG results showed a tendency towards saturation in the ^14C isotope ratio in the root system, the amount of radioactivity, and the distribution rate to the roots. Roots not only receive photosynthetic products from stems and leaves, but also excrete them out of the body as CO ₂ through respiration. It is known that the rate of root respiration increases under drought stress. (Refer to Crop Science Society of Japan Conference, p. 192 (2017)), it was suggested that the rate of ¹⁴ C supply from the shoots to the roots and the rate of ¹⁴ C excretion through respiration may have reached an equilibrium state. CRDS is a method for analyzing gases, and in the future, in addition to evaluations similar to those in this experiment, it is expected that new evaluation methods will be developed, such as evaluating the amount of ¹⁴ _CO2 in exhaled air from the root system. Ru.

From the above, we were able to obtain the temporal trends in the transport of photosynthetic products produced by leaves to roots and evaluate the differences due to the presence or absence of drought stress, demonstrating the dynamic evaluation of photosynthetic products in rice using the ^14C -CRDS analysis system. did. Based on the results obtained this time and the detection sensitivity evaluated ( ¹⁴ C/ ¹² C ~ ^2.0 It turned out that it is possible to evaluate based on quantity.

As an application of the ¹⁴ C analysis system based on mid-infrared CRDS ( ¹⁴ C-CRDS analysis system), we focused on the application of ¹⁴ C tracers in plant physiology, and conducted demonstration experiments to label rice samples with ¹⁴ C and evaluate its dynamics. , this method has utility in plant physiology. After constructing an introduction section for measuring plant samples and confirming its operation, basic experiments using ¹³ C-labeled corn samples showed that it was possible to measure carbon isotope ratios in plant samples. In addition, we constructed a ¹⁴ CO ₂ labeling system for plant samples, and labeled rice samples grown with and without drought stress with ¹⁴ C to determine the time period during which ¹⁴ C taken up from leaves through photosynthesis is transferred to roots. Obtained the latest trend. We were able to clearly evaluate the carbon dynamics of ^14C moving from leaves to roots, and found that the dynamics differed depending on the presence or absence of drought stress. At the same time, since it has a sensitivity several orders of magnitude higher than existing analytical methods, it is expected that similar evaluations can be made with a smaller amount of administered radioactivity and sample.
Future prospects include the development of a method for measuring exhaled ¹⁴ CO ₂ in roots, measurement of exudates from stems and leaves, dynamic evaluation of minute parts such as leaves and stems, main roots and lateral roots, and analysis of ¹⁴ C in metabolites. It is expected that this method will be applied to various analyzes that could not be realized due to the lack of suitable analytical methods.

1A, 1B Carbon isotope analyzer 10A, 10B Spectroscopic device 11 Optical resonator 12a, 12b Mirror 13 Piezo element 15 Photodetector 16 Cell 18 Vacuum device 19 Peltier element 20A, 20B Light generator 21 First optical fiber 22 Second optical fiber 23 Light source 24 Nonlinear optical crystal 25 Optical switch 26a to 26e Mirror 28 Beat signal measuring device 29 Optical branching device 30 Arithmetic device 40 Carbon dioxide isotope generator 80 14CO2 photosynthesis labeling system for plant samples 81 Vial 83 Sample bag
85 Desiccator 86 Circulation pump 88a, 88b Piping 86 Circulation pump 89a to 89c Valve

Claims (13)

¹⁴ _CO2 photosynthesis labeling system for plant samples;
A carbon isotope analyzer for analyzing plant samples,
The carbon isotope analyzer for plant sample analysis includes:
a carbon dioxide isotope generator comprising a combustion section that generates a gas containing ¹⁴ CO ₂ from ¹⁴ C;
a light generator;
An optical resonator having a pair of mirrors, a spectroscopic device including a photodetector that detects the intensity of transmitted light from the optical resonator ,
The ¹⁴ CO ₂ photosynthesis labeling system for plant samples includes :
¹⁴ A vial that generates CO ₂ gas,
A sample bag that accumulates the generated gas,
a desiccator into which the plant sample is introduced;
circulation pump,
a first pipe connecting the vial and the sample bag;
a second pipe connecting the sample bag and the desiccator;
a third pipe connecting the sample bag and the desiccator via the circulation pump;
has
The volume of the sample bag is small compared to the volume of the desiccator;
First, only the first pipe from the first pipe to the third pipe is opened to accumulate ^{the 14} CO ₂ gas generated in the vial in the sample bag,
After that, only the second pipe from the first pipe to the third pipe is opened to fill the desiccator with ¹⁴ CO ₂ gas,
Thereafter, the second pipe and the third pipe from the first pipe to the third pipe are opened to circulate gas between the sample bag and the desiccator using the circulation pump. The plant sample analysis system according to claim 1 , wherein the combustion section includes a combustion furnace arranged substantially perpendicular to the direction of gravity, a sample boat, a carrier gas supply tank, and a dehumidifier. . The plant sample according to claim 1 or ₂ , further comprising a carbon dioxide isotope trap provided with a cooling device for freezing the ¹⁴ CO2 , disposed between the carbon dioxide isotope generating device and the spectroscopic device . Analytical system. Claims 1 to 3 , wherein the light generating device includes a light source, an optical switch that controls on/off of light from the light source, and a mirror that reflects the light from the optical switch and sends the light back to the optical switch. The plant sample analysis system according to any one of the above. 5. The system for analyzing plant samples according to claim 4 , wherein the optical switch is an acousto-optic modulator. The light generating device includes a branching means for branching the light from the light source, a condensing lens for condensing the light from the branching means, and a condensing lens that reflects the light from the condensing lens to connect the condensing lens and the branching means. The system for analyzing plant samples according to claim 4 or claim 5 , comprising a mirror that sends light back to the light source via the mirror. The system for analyzing plant samples according to any one of claims 4 to 6, wherein the light source generates optical frequency comb light. The plant sample analysis system according to claim 4, wherein the light source is a fiber laser. The plant sample analysis system according to claim 4 , wherein the light source is a mid-infrared quantum cascade laser. The light source is
A mid-infrared quantum cascade laser as the main light source,
An optical comb source that generates an optical comb consisting of a bundle of light with a narrow linewidth in which the frequency range of one light is 4500 nm to 4800 nm, and a beat caused by the frequency difference between the light from the main light source and the light from the optical comb source. a beat signal measuring device comprising a photodetector for measuring a signal;
The plant sample analysis system according to any one of claims 4 to 6 , comprising: photosynthesizing a plant sample using a ¹⁴ CO photosynthesis labeling method of a plant _sample , and labeling the plant sample with ¹⁴ C;
producing ¹⁴ CO ₂ from ¹⁴ C in the plant sample;
filling the optical resonator with the ¹⁴ CO ₂ ;
irradiating the inside of the optical resonator with irradiation light having an absorption wavelength for the ¹⁴ CO ₂ ;
Measuring the intensity of transmitted light obtained when the ¹⁴ CO ₂ is irradiated with the irradiation light and resonated;
calculating ^{the 14} C concentration from the intensity of the transmitted light ,
The ¹⁴ CO ₂ photosynthesis labeling method of the plant sample includes :
_generating ¹⁴ CO2 gas in the vial ;
_accumulating the generated ¹⁴ CO2 gas in a sample bag;
A step in which a plant sample is introduced and a dessicator having a larger volume than the sample bag is filled with ¹⁴ CO ₂ gas from the sample bag;
A carbon isotope analysis method for plant sample analysis , comprising the step of circulating gas between the sample bag and the desiccator . Between the step of irradiating the irradiation light and the step of measuring the intensity of the transmitted light, light from the light source is introduced into the optical switch, and the light emitted from the optical switch is sent back to the optical switch to turn the light on and off. The carbon isotope analysis method for plant sample analysis according to claim 11 , further comprising the step of controlling. 13. According to claim 11 or 12 , as the irradiation light, an optical comb having a mid-infrared light frequency in a wavelength band of 4.5 μm to 4.8 μm is generated from a difference in frequency by passing a plurality of lights through a nonlinear optical crystal. carbon isotope analysis method for plant sample analysis.

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