JP2021032632A - Carbon isotope analyzer for plant sample analysis and carbon isotope analysis method for plant sample analysis using the same - Google Patents

Abstract

To provide a carbon isotope analyzer suitable for analysis of a plant sample with which, of a simple structure though, it is possible to analyze with good accuracy without performing a complex preprocess and a carbon isotope analysis method using this device, as well as provide a system for plant sample analysis equipped with the carbon isotope analyzer and a 14CO2 photosynthesis labeling system.SOLUTION: Provided is a carbon isotope analyzer for plant sample analysis comprising: a carbon dioxide isotope generator equipped with a combustion unit for generating a gas that includes a carbon dioxide isotope from a carbon isotope; a light generator; and a spectrometer equipped with an optical resonator having a pair of mirrors and a photodetector for detecting the strength of transmitted light from the optical resonator.SELECTED DRAWING: Figure 2

Description

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

Carbon isotopes have been widely applied to various fields such as environmental dynamics evaluation based on the carbon cycle and empirical research on history by dating. Although the carbon isotopes differ slightly depending on the region and environment, the stable isotopes ¹² C and ¹³ C are 98.89% and 1.11%, respectively, and the radioactive isotope ¹⁴ C is 1 × 10 ^-10 % natural. Exists in. Since isotopes have the same chemical behavior except for the difference in weight, the concentration of isotopes with a low abundance ratio is increased by artificial manipulation, and accurate measurement is performed for various reaction processes. Observation becomes possible.

¹⁴ C Tracer is used as a useful tool for evaluating various dynamics important in plant physiology in plants, especially crops such as grains and vegetables, in addition to medical and pharmaceutical applications. For example, a research group of M. Calvin et al. Of the University of California, USA, ^{absorbed 14} CO ₂ into plants by photosynthesis and elucidated the scientific reaction process (see, for example, Non-Patent Document 1). The process elucidated by this study is called the Calvin cycle, and won the Nobel Prize in Chemistry in 1961.
In such ^14C tracer application in plant physiology, a method basically based on radiation measurement has been used. For example, a research group of the University of Tokyo, Tanoi et al. Has developed a system for imaging (live imaging) β-rays derived from ^{14 C administered to plants without stopping biological activity (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 the dynamics, ^{the β-ray energy from 14} C is weak and is absorbed in the plant body, resulting in low measurement sensitivity. Quantitative analysis is difficult.

Conventionally proposed ¹⁴ C 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.

Japanese 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 the LSC is a relatively small tabletop-sized device, simple and quick analysis is possible. However, when measuring a plant sample with LSC, if the plant sample is put into an organic solvent without pretreatment, there is a problem (color quenching) that the solution is colored and the count of scintillation light is reduced. Moreover, since ^{the detection limit concentration of 14} C of LSC is as high as 10 dpm / mL, the detection sensitivity is limited, and it is difficult to measure with a small amount of sample.

On the other hand, AMS has a ^{low detection limit concentration of 14} C of 0.001 dpm / mL, which ^{is 1000 times or more lower than the detection limit concentration of 14} C of LSC, so that it has high detection sensitivity and can withstand use, but the device is large and expensive. Therefore, its use was restricted. Although some techniques have been proposed as means for solving this problem of AMS (see, for example, Non-Patent Document 3 and Patent Document 1), Cavity Ring-Down Spectroscopy (hereinafter referred to as "CRDS"). ^{Although 14} C analysis by (also called) was demonstrated, the 4.5 μm band laser light generator used had an extremely complicated structure, so a simpler and easier-to-use ¹⁴ C analyzer and analysis method were required. Was being done. Moreover, the structure was not conscious of the analysis of plant samples.
From the above, there has been a demand for a carbon isotope analyzer suitable for analysis of plant samples and a carbon isotope analysis method using the same, which has a simple structure and can be analyzed with high sensitivity without performing complicated pretreatment. ..

The present invention relates to the following contents.
[1] From a carbon dioxide isotope generator including a combustion unit that generates a gas containing a carbon dioxide isotope from a carbon isotope, a light generator, an optical resonator having a pair of mirrors, and the optical resonator. A spectroscopic device including a light detector for detecting the intensity of transmitted light, and a carbon isotope analyzer for plant sample analysis.
[2] The carbon isotope analyzer for plant sample analysis according to [1], wherein the carbon isotope is radiocarbon isotope ¹⁴ C and the carbon dioxide isotope is radiocarbon dioxide isotope ¹⁴ CO _2.
[3] The combustion unit includes a combustion furnace arranged substantially perpendicular to the direction of gravity, a sample boat, a carrier gas supply tank, and a dehumidifying device, according to [1] or [2]. Carbon isotope analyzer for plant sample analysis.
[4] Any of [1] to [3] further comprising a carbon dioxide isotope trap having a carbon dioxide isotope freezing cooling device arranged between the carbon dioxide isotope generator and the spectroscopic device. The carbon isotope analyzer for plant sample analysis according to one.
[5] The light generator includes a light source, an optical switch for controlling the 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 [1] to [4]. ]. The carbon isotope analyzer for plant sample analysis according to any one of the above.
[6] The carbon isotope analyzer for carbon dynamics analysis in a plant material according to [5], wherein the optical switch is an acousto-optic modulator.
[7] The light generator includes a branching means for branching light from a light source, a condensing lens for condensing light from the branching means, and a condensing lens and branching means for reflecting light from the condensing lens. The carbon isotope analyzer for plant sample analysis according to any one of [1] to [6], which comprises a mirror that sends light back to a light source.
[8] The carbon isotope analyzer for plant sample analysis according to any one of [1] to [7], wherein the light source is an optical frequency comb light.
[9] The carbon isotope analyzer for plant sample analysis according to any one of [1] to [8], wherein the light source is a fiber laser.
[10] The carbon isotope analyzer for plant sample analysis according to any one of [1] to [7], wherein the light source is a mid-infrared quantum cascade laser.
[11] The light source is a mid-infrared quantum cascade laser as a main light source, and an optical comb source and a main light source that generate an optical comb consisting of a bundle of light having a narrow line width in which a frequency region of one light is 4500 nm to 4800 nm. The invention according to any one of [1] to [7], comprising a beat signal measuring device including an optical detector for measuring a beat signal generated by a frequency difference between light from and an optical comb source. Carbon isotope analyzer for plant sample analysis.
[12] The carbon isotope analyzer for plant sample analysis according to any one of [1] to [11], ^{a vial for generating 14} CO ₂ gas, a sample bag for accumulating the generated gas, and a plant sample. A system for plant sample analysis including a ¹⁴ CO ₂ photosynthetic labeling unit for plant samples equipped with a desiccator and a circulation pump to be introduced.
[13] ¹⁴ A vial for generating CO ₂ gas, a sample bag for accumulating the generated gas, and a sample bag.
^{A 14} CO ₂ photosynthetic labeling system for plant samples, including a desiccator into which the plant sample is introduced and a circulation pump.
[14] ¹⁴ A step of photosynthesizing a plant sample in an environment containing CO ₂ gas, a step of producing a carbon dioxide isotope from a carbon isotope in the plant sample, and a step of filling the photoresonator with the carbon dioxide isotope. A step of irradiating the photosynthetic device with irradiation light having an absorption wavelength for the carbon dioxide isotope and a step of measuring the intensity of transmitted light obtained when the carbon dioxide isotope is irradiated with the irradiation light and resonated. A carbon isotope analysis method for plant sample analysis, which comprises a step of calculating a carbon isotope concentration from the intensity of transmitted light.
[15] Between the step of irradiating the irradiation light and the step 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 [14], further comprising a step of controlling.
[16] As the irradiation light, by passing a plurality of lights through the nonlinear optical crystal, an optical comb having a wavelength in the mid-infrared region of 4.5 μm to 4.8 μm is generated from the difference in frequency, [14] or [ 15] The carbon isotope analysis method for plant sample analysis.

According to the present invention, there is provided a carbon isotope analyzer suitable for analysis of a plant sample, which has a simple structure and can be analyzed with high sensitivity without performing complicated pretreatment, and a carbon isotope analysis method using the same. Will be done. Further, a system for plant sample analysis including the above carbon isotope analyzer and a ¹⁴ CO _{2 photosynthesis labeling system is provided.}

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 a 4.5 μm band absorption spectrum ^{of 14} CO _{2 and a competing gas.} FIG. 4 is a diagram showing the relationship ^{of CO 2} absorption with respect to the time from each sample introduction of (a) standard glucose aqueous solution, (b) solid standard glucose, and (c) corn foliage 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 amounts Δβ ^{of 13} CO ₂ and ¹⁴ CO _{2 in CRDS.} FIG. 7 is a conceptual diagram (partially cutaway diagram) of a specific embodiment of the optical resonator. 8A and 8B are schematic views of one aspect of the light generator, respectively. FIG. 9A is a schematic view of a light generator provided with a double path. FIG. 10 is a schematic view of the periphery of the optical switch in the light generator. 11A and 11B are the residuals in the fitting by the attenuation function for obtaining the ringdown signal acquired by a single pass and the attenuation rate thereof. 12A and 12B are the residuals in the fitting by the attenuation function for obtaining the ringdown signal acquired by the double path and the attenuation rate thereof. FIG. 13 shows the measurement of the sum of squares of the residuals in the fitting for each ringdown signal for a large number of ringdown signals (variation of the sum of squares of the residuals). FIG. 14 is a schematic view of the carbon isotope analyzer 1B according to the second aspect. FIG. 15 is a diagram showing the relationship between the absorption wavelength and the absorption intensity of ^{the analytical 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 views of a second aspect of the carbon isotope analysis method. Is 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. 19A and 19B are diagrams showing the acquired spectra, respectively. FIG. 20A is a diagram in which the vertical axis is ^{standardized with an intensity of 12} CO ₂ from FIG. 19A, and FIG. 20B is a diagram in which the vertical axis is standardized with an intensity of ¹² CO _{2 from FIG. 19B.} FIG. 21 is a plot of d13C measured for each sample based on the results shown in Table 1. 22A and 22B are diagrams ^{showing the results of the 14} C labeled / unlabeled foliage sample when the absorption spectrum was acquired by the ^{14 C-CRDS analysis system.} 23A and 23B are diagrams showing absorption spectra of the root system and foliage of the same individual sampled immediately after the PEG group for the root system and foliage. ^{24A and 24B are diagrams showing 14} C isotope ratios for the foliage and root system with respect to the elapsed time from each label. ^{25A and 25B are diagrams showing a 14} C time change in the root system and the foliage as the amount of radioactivity in the sample with respect to the elapsed time from the label. FIG. 26 is a diagram showing the result of dividing ^{the 14 C isotope ratio of the root system by the 14} C isotope ratio of the foliage based on the result of FIG. 25. FIG. 27 is a diagram showing the distribution rate for each elapsed time from the root system (root) to the foliage (shoot), which was evaluated from the result of FIG. 25.

Hereinafter, the present invention will be described with reference to embodiments, but the present invention is not limited to the following embodiments. In the figure, those having the same function or similar functions are designated by the same or similar reference numerals and the description thereof will be omitted. However, the drawings are schematic. Therefore, the specific dimensions and the like should be determined in light of the following explanations. In addition, it goes without saying that the drawings include parts having different dimensional relationships and ratios from each other.

[First aspect of carbon isotope analyzer for plant sample analysis]
As an analysis target, a carbon isotope analyzer will be described by taking ^{radioactive isotope 14C, which is a carbon isotope, as an example.} The light having an absorption wavelength of the carbon dioxide isotope ¹⁴ CO ₂ ^{produced from the radioisotope 14} C is light in the 4.5 μm band. Although the details will be described later, high sensitivity can be realized by the selectivity of the absorption line of the substance to be measured, the light generator, and the optical resonator mode.

As used herein, the term "carbon isotope" means stable carbon isotopes ¹² C, ¹³ C, and radiocarbon isotopes ¹⁴ C, unless otherwise specified. Also, when simply displayed as the element symbol "C", it means a carbon isotope mixture in the natural abundance ratio.
^{There are 16} O, ¹⁷ O and ¹⁸ O stable isotopes of oxygen, but when the element symbol "O" is displayed, it means an oxygen isotope mixture in the natural abundance ratio.
"Carbon dioxide isotope" means ¹² CO ₂ , ¹³ CO ₂ and ¹⁴ CO ₂ unless otherwise noted. When simply _{displayed as "CO 2} ", it means a carbon dioxide molecule composed of carbon and oxygen isotopes having a natural abundance ratio.

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 a computing device 30.
The carbon dioxide isotope generation device 40 includes a combustion unit that generates a gas containing a carbon dioxide isotope from a carbon isotope, and a carbon dioxide isotope purification unit.
The spectroscope 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>
As the carbon dioxide isotope generation device 40, various devices can be used without particular limitation as long as the carbon isotope can be converted into the carbon dioxide isotope. The carbon dioxide isotope generator 40 preferably has a function of oxidizing the sample and converting carbon contained in the sample into carbon dioxide. For example, carbon dioxide such as a total organic carbon (hereinafter referred to as "TOC") generator, a sample gas generator for gas chromatography, a sample gas generator for combustion ion chromatography, and an elemental analyzer (EA). A carbon generator (G) 41 can be used. Considering the combustion of the plant sample, it is preferable to use one in which the combustion portion is arranged substantially perpendicular to the direction of gravity, as described later.

^{Figure 3 shows 14} CO ₂ ^{and 13} CO competing gas 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%. The 4.5 μm band absorption spectra of ₂ , CO, and N _{2 O are shown.}
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 produced. However, 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, and therefore compete with the absorption spectrum in the 4.5 μm band of ¹⁴ CO _2. Therefore, it is preferable to remove _{CO and N 2} O in order to improve the analysis sensitivity.
Examples of the method for removing _{CO and N 2} O include a method for collecting and separating ¹⁴ CO _{2 as follows.} Further, the oxidation catalyst or platinum catalyst, CO, a method of removing and reducing the N 2 _O, and the combination with the collection and separation methods used.

^{(I) Collection / separation of 14} CO ₂ by a heat desorption column The carbon dioxide isotope generator can be provided with a combustion unit and a carbon dioxide isotope purification unit. The combustion unit may include a combustion pipe and a heating unit that enables the combustion pipe to be heated. It is preferable that the combustion tube is made of heat-resistant glass (quartz glass or the like) so that the sample can be accommodated inside, and a sample introduction port is formed in a part of the combustion tube. In addition to the sample introduction port, the combustion pipe may form a carrier gas introduction port so that the carrier gas can be introduced into the combustion pipe. In addition to the mode in which the sample introduction port or the like is provided in a part of the combustion pipe, a sample introduction part is formed at one end of the combustion pipe by a member different from the combustion pipe, and the sample introduction part or the carrier gas is introduced into the sample introduction part. It may be configured to form a mouth.
Examples of the heating unit include an electric furnace such as a tubular electric furnace in which the combustion pipe can be arranged inside and the combustion pipe can be heated. An example of a tubular electric furnace is ARF-30M (Asahi Rika Seisakusho).
Further, it is preferable that the combustion pipe is provided with an oxidizing portion and / or a reducing portion filled with at least one kind of catalyst on the downstream side of the carrier gas flow path. The oxidizing part and / or the reducing part may be provided at one end of the combustion pipe or may be provided as a separate member. Examples of the catalyst to be filled in the oxidized portion include copper oxide and a mixture of silver and cobalt oxide. In the oxidizing part, it can be expected _{that H 2} and CO generated by the combustion of the sample are oxidized _{to H 2} O and CO _2. Examples of the catalyst to be filled in the reducing portion include reduced copper and platinum catalysts. Nitrogen oxides (NO _X ) containing N ₂ O can be expected to be reduced to N _{2 in the reducing section.}
As the carbon dioxide isotope purification unit _{, a heat desorption column (CO 2} collection column) such as that used in gas chromatography (GC) for ¹⁴ CO ₂ in a gas generated by combustion of a biological sample can be used. .. As a result, _{the influence of CO and N 2} O can be reduced or eliminated at the stage of detecting ¹⁴ CO _2. Also by CO ₂ gas is trapped Temporary containing ¹⁴ CO ₂ in the GC ^column, since the 14 CO ₂ concentration is ^expected, it is expected to improve the partial pressure of 14 CO _2.
(Ii) Trap of ¹⁴ CO ₂ ^{by 14} CO ₂ ^{adsorbent, separation of 14} CO ₂ by re-emission The carbon dioxide isotope generator 40b preferably includes a combustion unit and a carbon dioxide isotope purification unit. The combustion unit can be configured in the same manner as described above.
As the carbon dioxide isotope purification unit, a ¹⁴ CO ₂ adsorbent such as soda lime or calcium hydroxide can be used. As a result, the problem of contaminating gas can be solved by isolating ¹⁴ CO _{2 in the form of carbonate.} ^{Since it retains 14} CO ₂ as a carbonate, it is also possible to temporarily store the sample. Phosphoric acid can be used for re-release.
By providing either (i), (ii), or both configurations, the contaminating gas can be removed.
(Iii) ¹⁴ CO ₂ concentration (separation)
^{The 14} CO ₂ generated by the combustion of the biological sample diffuses in the pipe. Therefore, the detection sensitivity (strength) may be improved by adsorbing ¹⁴ CO _{2 on the adsorbent and concentrating it.} ^{Separation of 14} CO ₂ from CO and N ₂ O can be expected by such concentration.

FIG. 2 is a conceptual diagram of one aspect 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 of the screen), a sample boat 40b, a carrier gas supply tank 40c, and a dehumidifying device. It includes 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. Various dehumidifying devices 40d can be used without particular limitation, and for example, a membrane dryer can be used.
Measurement is started by placing the samples in the sample boat and inserting them into the combustion furnace. _{The CO 2} gas generated from the combustion furnace is carried by the carrier gas (pure oxygen), passes through the dehumidifying device, and then is introduced into the optical resonator 11.
The EA combustion furnace is arranged vertically, and the sample is burned by free-falling from the upper sample port to the lower furnace. Therefore, it is necessary to insert a sample such as a plant sample, which is likely to dissipate to the surroundings, into a tin capsule (melting point: about 230 ° C), which has a melting point sufficiently lower than the furnace temperature (900 ° C), like the liquid sample. There is. In plant measurement, it is often cut and powdered to average the differences between parts, making it difficult to reliably insert all weighed plant powder into relatively soft tin capsules. Yes, skillful skills are required. However, by configuring the carbon dioxide isotope generator 40A in FIG. 2, the work load is reduced and the certainty of sample introduction is improved.

[Confirmation experiment]
A major difference between the carbon dioxide isotope generator 40A and EA is the absence of a heat adsorption / desorption column. However, since the _{CO 2} trap is introduced _{as a CO 2} enrichment mechanism in the _{system, CO 2} is generated in a time pulse, so that there is no big hindrance to the measurement. The following experiments were conducted to show that the measurement was not hindered.
A device as shown in FIGS. 1 and 2 was prepared, and the wavelength of the DFB-QCL was fixed to 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 foliage powder sample were introduced. Then, _{the time profile in which CO 2} was generated and reached the CRDS analysis cell was evaluated. At this time, the CO ₂ trap system was introduced, but it was not immersed in liquid nitrogen. In addition, the timing at which the sample was inserted into the furnace was acquired as a trigger for opening and closing the valve instead of the temperature rise of the column, and used as the start time. The obtained results are shown in FIG. In the figure, the horizontal axis is the time from sample introduction, and the vertical axis is 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 in all the samples. On the other hand, there was a difference in the arrival timing between the liquid and the solid for about 10 seconds, and even in the same solid, the peak shape was different between the glucose and the corn foliage. _{It is considered that this is because the CO 2} generation time profile in the combustion furnace differs depending on the composition and shape (particle size, etc.) of the sample and the difference in heat transfer. Such a difference in timing and shape is _{sufficiently small for the CO 2} trap in the subsequent stage of the solid sample combustion device, and does not have a large effect on the measurement. In addition, the peak width of the time profile measured by the CRDS analysis cell was wider than that of EA.
This is due to the absence of the heat absorption / desorption column, and it is appropriate that the temporal distribution of combustion in the furnace is observed as it is. From the above, it was clarified that _{CO 2} ^{in a plant sample can be analyzed by the 14} CCRDS analysis system developed so far by connecting a solid sample combustion device.

<Carbon dioxide isotope trap>
As shown in FIG. 2, the carbon dioxide isotope trap 60 is arranged on the gas supply pipe 69a for sending the carbon dioxide isotope from the carbon dioxide isotope generator 40 to the spectroscopic device 10 and on the upstream side of the gas supply pipe 69. A valve 66a, a U-shaped trap pipe 61, a valve 66b arranged on the downstream side of the gas supply pipe 69, and a gas supply pipe 69b branched from the gas supply pipe 69a by the valve 66a and communicated with the optical resonator 11. , A pump P for making the gas supply pipe 69a and the resonator 11 negative pressure, a valve 66c arranged on the gas supply pipe 69b between the resonator 11 and the pump P, and a trap pipe 61 for cooling. It is provided with a dewar bottle 63 that can be filled with liquid nitrogen 65.
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.
The 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.

<Spectroscopic 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 is arranged so that the cylindrical main body in which the carbon dioxide isotope to be analyzed is sealed and the concave surface face each other at one end and the other end in the longitudinal direction inside the main body. It includes a pair of mirrors 12a and 12b having high reflectance, a piezo element 13 arranged at the other end of the main body for adjusting the distance between the mirrors 12a and 12b, 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 adjusting port for adjusting the air pressure in the main body on the side of the main body. The reflectance of the pair of mirrors 12a and 12b is preferably 99% or more, more preferably 99.99% or more.
When the laser beam is incident on the inside 11 of the optical resonator and confined, the laser beam repeats multiple reflections on the order of several thousand times to 10,000 times while outputting light having 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 enclosed inside the optical resonator is extremely small.

5A and 5B are diagrams showing the principle of high-speed scanning type cavity ring-down 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 and the non-resonant condition is set, the signal cannot be detected due to the interference effect of light. That is, by quickly changing the optical resonator length from resonance to non-resonance conditions, an exponential attenuation signal [Ringdown signal] as shown in FIG. 5A can be observed. As another method of observing the ringdown signal, a method of quickly blocking the input laser light with an optical switch can be exemplified.
When the inside of the optical resonator is not filled with an absorbent substance, the transmitted time-dependent ringdown signal has a curved line as shown by the dotted line in FIG. 5B. On the other hand, when the optical resonator is filled with an absorptive substance, as shown by the solid line in FIG. 8B, the laser beam is absorbed each time it reciprocates in the optical resonator, so that the light attenuation time is shortened. Since the decay time of this light depends on the concentration of the light-absorbing substance in the optical cavity and the wavelength of the incident laser light, the absolute concentration of the absorbing substance can be calculated by applying Beer-Lambert's law ii. .. Further, the concentration of the absorbent substance in the optical cavity can be measured by measuring the amount of change in the attenuation rate (ring down rate) which is proportional to the concentration of the absorbent substance in the optical cavity.

After detecting the transmitted light leaked from the optical resonator with a photodetector and calculating the ¹⁴ CO ₂ ^{concentration using an arithmetic unit, the 14} C concentration can be calculated from ^{the 14} CO _{2 concentration.}

The intervals between the mirrors 12a and 12b of the optical resonator 11, the radius of curvature of the mirrors 12a and 12b, the length and width in the longitudinal direction of the main body, and the like are preferably changed according to 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 carbon dioxide isotope ¹⁴ CO _2, a long resonator length is effective in securing the optical path length, but as the resonator length increases, the volume of the gas cell increases and the required sample amount increases, so resonance occurs. The instrument length is preferably between 10 cm and 60 cm. Further, the radius of curvature of the mirrors 12a and 12b is preferably the same as or longer than the cavity length.
By driving the piezo element 13, the mirror spacing can be adjusted on the order of several micrometers to several tens of micrometers as an example. Fine adjustment by the piezo element 13 can also be performed in order to create the optimum resonance condition.
As the pair of mirrors 12a and 12b, a pair of concave mirrors has been illustrated and described, but if a sufficient optical path can be obtained, a combination of a concave mirror and a plane mirror or a combination of plane mirrors can be used. It doesn't matter if there is.
_{Sapphire glass, CaF 2} , and ZnSe can be used as the materials constituting the mirrors 12a and 12b.
The cell 16 filled with the gas to be analyzed preferably has a smaller volume. This is because the resonance effect of light can be effectively obtained even with a small number of analytical samples. The volume of the cell 16 can be exemplified by 8 mL to 1000 mL. As the cell volume, for example, a preferable volume can be appropriately selected according to the amount of ¹⁴ C source that can be used for measurement, and for a ¹⁴ C source that can be obtained in large quantities such as urine, 80 mL to 120 mL of cells are preferable, and blood or cells are suitable. ^{For 14} C sources with limited availability, such as tears, 8 mL-12 mL cells are suitable.

Evaluation of Stability Conditions of Optical Resonator In ^{order to evaluate the amount of 14} CO ₂ absorbed and the detection limit in CRDS, calculations based on spectroscopic data were performed. ^For spectroscopic data on 12 CO ₂ , ¹³ CO _2, etc., the atmospheric absorption line database (HITRAN) was used, and for 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 ^{) due to absorption of 14} CO _{2 is} ¹⁴ CO ₂ can be expressed as follows by the light absorption cross section σ ₁₄ , the molecular number density N, and the light velocity c.
Δβ = σ ₁₄ (λ, T, P) N (T, P, X ₁₄ ) c
(In the equation, σ ₁₄ , N is a function of the laser light wavelength λ, the temperature T, the pressure P, and the X ₁₄ = ¹⁴ C / ^Total C ratio.)
FIG. 6 is a diagram showing the temperature dependence of Δβ due to the absorption of ¹³ CO ₂ and ¹⁴ CO _{2 obtained by calculation.} From FIG. ^{9, 14} C / ^Total C ^{10 -10,} 10 ^-11, in ^{10 -12,} for a equal to or absorption by ¹³ CO ₂ at room temperature 300K exceeds the absorption of ¹⁴ CO _2, the cooling I found that I needed to do it.
On the other hand, it can be seen that if the variation in the ringdown rate, which is a noise component derived from the optical resonator, Δβ ₀ to 10 to ¹ s ^-1 , can be realized, ^{the measurement of 14} C / ^Total C ratio to 10 to ^{11 can be realized.} From this, it became clear that cooling of about -40 degrees Celsius is required as the temperature at the time of analysis.
For example, when the ¹⁴ C / ^Total C and ^10-11 as the lower limit of quantification, increase of CO ₂ gas partial pressure by concentration of CO ₂ gas (e.g. 20%), suggesting that it is necessary and the temperature ..
The cooling device and the cooling temperature will be described in more detail in the column of the second aspect of the carbon isotope analyzer described later.

Although the optical resonator 11 has been described, FIG. 7 shows a conceptual diagram (partially cutaway diagram) of a specific embodiment of the optical resonator. As shown in FIG. 7, the optical resonator 51 is arranged at both ends of a cylindrical heat insulating chamber 58 as a vacuum device, a measuring gas cell 56 arranged in the heat insulating chamber 58, and a measuring gas cell 56. Cools the pair of high-reflectivity mirrors 52, the mirror drive mechanism 55 arranged at one end of the measurement gas cell 56, the ring piezo actuator 53 arranged at the other end of the measurement gas cell 56, and the measurement gas cell 56. A Peltier element 59 is provided, and a water-cooled heat sink 54 having a cooling pipe 54a connected to a circulation cooler (not shown) is provided.

<Arithmetic logic unit>
The arithmetic unit 30 is not particularly limited as long as it can measure the concentration of the absorbent substance in the optical resonator from the above-mentioned decay time and ring down rate and can measure the carbon isotope concentration from the concentration of the absorbent substance. The device can be used.
The arithmetic control unit 31 may be configured by arithmetic means or the like used in a normal computer system such as a CPU. Examples of the input device 32 include a pointing device such as a keyboard and 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, a storage device such as a ROM, RAM, or magnetic disk can be used.

<Light generator>
As the light source 23, various light sources can be used without particular limitation, but 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 that a nonlinear optical effect easily occurs, and light in the 4.5 μm band, which is the absorption wavelength of ^{the radioactive carbon dioxide isotope 14} CO _2. Can be easily generated. Further, since a comb-shaped bundle of light having a uniform wavelength width of each wavelength (optical frequency comb, hereinafter also referred to as “optical comb”) can be obtained, the fluctuation of the oscillation wavelength can be made negligibly small. When a continuous oscillation generator is used as the light source, the oscillation wavelength fluctuates, so it is necessary to measure the oscillation wavelength fluctuation with an optical comb or the like.
As the light source 23, for example, a solid-state laser, a semiconductor laser, or a fiber laser that outputs a short pulse by mode-locking can be used. Of these, it is preferable to use a fiber laser. This is because the fiber laser is a practical light source that is compact and has excellent environmental stability.
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 the economical point of view, it is preferable to use a general-purpose Er-based fiber laser, and from the viewpoint of increasing the light intensity, it is preferable to use a Yb-based fiber laser.

FIG. 8A shows one aspect (difference frequency mixing) of the light generator. The light generator 20A is a first 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 merges at a confluence point on the downstream side of the first optical fiber 21. A second optical fiber 22 that transmits light having a wavelength longer than that of the optical fiber 21 and a nonlinear optical crystal 24 that generates light having an absorption wavelength of carbon dioxide isotope from a difference in frequency by passing a plurality of light having different frequencies. 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 merges on the downstream side of the first optical fiber 21. Can be used. As the first optical fiber 21, one connected from the light source to the optical resonator can be used. Further, in each optical fiber, a plurality of optical components and a plurality of types of optical fibers can be arranged on each path.
As the first optical fiber 21, it is preferable to use an optical fiber capable of transmitting the generated high-intensity ultrashort pulsed light without deteriorating the characteristics. Specifically, dispersion compensating fiber (DCF), double clad fiber and the like can be included. As the material, it is preferable to use a fiber made of fused silica.
As the second optical fiber 22, it is preferable to use an optical fiber that can efficiently generate ultrashort pulsed light on the desired long wavelength side and transmit the generated high-intensity ultrashort pulsed light without deteriorating the characteristics. Specifically, a polarization-retaining fiber, a single-mode fiber, a photonic crystal fiber, a photonic band gap fiber, and the like can be included. It is preferable to use an optical fiber having a length of several meters to several hundreds of meters according to the amount of wavelength shift. As the material, it is preferable to use a fiber made of fused silica.

The nonlinear optical crystal 24 is appropriately selected according to the incident light and the emitted light, but in the case of this embodiment, it is said that light having a wavelength of about 4.5 μm band is generated from each incident light. From the viewpoint, for example, PPMgSLT (periodically poled MgO-doped Stoichiometric Lithium Tantalate (LiTaO ₃ )) crystal, PPLN (periodically poled Lithium Niobate) crystal, or GaSe (Gallium selenide) crystal can be used. Further, since one fiber laser light source is used, the fluctuation of the optical frequency can be canceled in the difference frequency mixing as described later.
The nonlinear optical crystal 24 preferably has a length in the irradiation direction (longitudinal direction) of 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"), a plurality of lights having different wavelengths (frequency) transmitted by the first and second optical fibers 21 and 22 are passed through a nonlinear optical crystal. From the difference in frequency, light corresponding to the difference frequency can be obtained. That is, in the case of this embodiment, _{two lights having wavelengths λ 1} and λ ₂ are generated from one light source 23, and the two lights are passed through the nonlinear optical crystal, so that the carbon dioxide isotope is generated from the difference in frequency. It can generate light of absorption wavelengths of the body. The conversion efficiency of a DFG using a nonlinear optical crystal depends on the photon density of the light sources _{having a plurality of wavelengths (λ 1} , λ ₂ , ... λ _x). Therefore, it is possible to generate light of a different frequency by DFG from one pulse laser light source.
Thus the light of 4.5μm band obtained by the optical comb comprising a plurality of frequencies of light for one pulse is regular frequency interval _{f r} (mode) (Frequency _{f = f ceo + N · f} r, N: Mode Number). In order to perform CRDS using the optical comb, it is necessary to introduce the light in the absorption band of the analysis target into the optical resonator including the analysis target. _{In the generated optical comb, f ceo} is canceled in the process of mixing the difference frequency, _{and f ceo} becomes 0.

In the case of the carbon isotope analyzer devised by I. Galli et al. Of Non-Patent Document 1, two types of laser devices (Nd: YAG laser and external-cavity diode laser (ECDL)) with different wavelengths are prepared and lasers are prepared. Irradiation light having an absorption wavelength of carbon dioxide isotope was generated from the difference in the frequency of light. Since both are continuously oscillating lasers and the intensity of ECDL is low, 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 of both is put into the optical resonator. , It was necessary to increase the photon density. Further, 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 equipment was large and the operation was complicated, such as the need to control the resonator that performs these operations. On the other hand, since the light generator according to the embodiment of the present invention is composed of one fiber laser light source, an optical fiber of several meters, and a nonlinear optical crystal, it is compact, easy to transport, and easy to operate. .. Further, since a plurality of lights are generated from one light source, the fluctuation width and the fluctuation timing of each light are the same. Therefore, the fluctuation of the optical frequency can be easily canceled by performing the difference frequency mixing without using the control device.
An optical system that transmits laser light into the air from the confluence of the first optical fiber and the second optical fiber to the optical resonator, and collects and / or magnifies the laser light by a lens as necessary. An optical transmission device including the above may be constructed.

In this analysis, it is ^{sufficient that the optical comb is obtained in a range covering the wavelength region used in the 14C} analysis. Therefore, the present inventors, the narrower the oscillation spectrum of the optical comb light source, the higher the output. I paid attention to the fact that the light of the frequency comb was obtained. When the oscillation spectrum is narrow, amplification by amplifiers having different bands or long nonlinear optical crystals can be used. Therefore, as a result of studies by the present inventors, in the generation of optical combs using the differential frequency mixing method, (a) a plurality of lights having different frequencies are generated from one light source, and (b) a plurality of obtained lights are obtained. (C) By passing multiple lights through a nonlinear optical crystal that is longer than the conventional nonlinear optical crystal, the absorption wavelength of the carbon dioxide isotope is determined by the difference in frequency. The idea was to generate high-power irradiation light with. The present invention has been completed based on the above findings. In the conventional differential frequency mixing method, there has been no report that the intensity of light is amplified by using a plurality of amplifiers having different bands, or that high-power irradiation light can be obtained by using a long crystal.

When the light absorption of the light absorbing substance has a high absorption line intensity and the light intensity of the irradiation light is also high, the lower level corresponding to the light absorption is remarkably reduced, and the effective light absorption amount seems 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 cavity is irradiated with light in the 4.5 μm band having a large absorption line intensity, the initial attenuation signal (ring down signal) obtained is initially. Since the light intensity stored in the optical resonator is high, the saturation effect is large, and then, as the attenuation progresses, the light intensity stored in the optical resonator gradually decreases, so that the saturation effect becomes smaller. Therefore, the attenuation 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 by the sample and the background attenuation rate can be evaluated independently, so that it is affected by fluctuations in the background attenuation rate such as the parasitic etalon effect. Since the attenuation rate of the sample can be obtained without any problem and the saturation effect of ¹⁴ CO ₂ ^{is larger than that of the contaminating gas, the 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 generator of the present invention can generate irradiation light having high light intensity, it is expected that the analysis sensitivity will be improved when used for carbon isotope analysis.

FIG. 8B shows one aspect (cat eye) of the light generator. FIG. 2 is a conceptual diagram of a carbon dioxide isotope generator and a carbon dioxide isotope trap. The light generator 20B is a first 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 merges at a confluence on the downstream side of the first optical fiber 21. A second optical fiber 22 that transmits light having a wavelength longer than that of the optical fiber 21, and a nonlinear optical crystal 24 that generates light having an absorption wavelength of carbon dioxide isotope from a difference in frequency by passing a plurality of lights having different frequencies. It includes an optical switch 25 that controls the on / off of 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 path>
FIG. 9A is a schematic view of the light generator. The present inventors conducted further studies in order to further improve the analysis accuracy of the carbon isotope analyzer, and found that the attenuation rate was lower than the expected performance (on-off ratio) of the optical switch. It was found that there was an error (residual in fitting by the attenuation function to obtain the attenuation rate of the ring-down signal). However, no simple and effective on / off control mechanism or method was found. Therefore, it has been required to eliminate the residual in the fitting of the ringdown signal and improve the analysis accuracy by improving the performance (on / off ratio) of the optical switch.
According to the present invention, there is provided a light generator having a small residual in fitting of a ring-down signal, a radiocarbon isotope analyzer using the same, and a radiocarbon isotope analysis method.
The light generator 20C includes a light source 23, an optical switch 25 that controls on / off of light from the light source 23, and mirrors 26a and 26b that reflect the light from the light switch 25 and send the light back to the light switch 25. The optical path 21 is not particularly limited, but for example, an optical fiber can be arranged.
The light generator 20C further includes mirrors 26c, 26d, 26e that introduce the light from the optical switch 25 into the light spectroscope 10A.
As the light source 23, various light sources can be used without particular limitation. Details will be described later.
Various optical switches 25 can be used without particular limitation, but it is preferable to use an acousto-optic modulator (hereinafter, also referred to as “AOM”) including an optical crystal 25a and a piezoelectric element 25b. ..

FIG. 10 is a schematic view of the periphery of the optical switch in the light generator. As shown in path 1 of FIG. 10, when the piezoelectric element 25b of the AOM is operated, the acoustic wave propagates in the optical crystal 25a. As a result, a periodic refractive index distribution is created in the optical crystal, and the incident light is diffracted to control the on / off of the light from the light source 23. However, even if the light emission is controlled off, there is a problem that the slightly leaked uncontrolled light causes an error in the ringdown signal. Therefore, in order to solve the above problems, the present inventors have completed a light generator having a double path in which mirrors 26a and 26b are arranged.

Next, the operation and action effect of the light generator will be described. (A) As shown in path 1 (P1) of FIG. 10, light from the light source 23 is sent to the optical switch 25 and on / off control is performed using the piezoelectric element 25b. After that, the light leaked from the (b) optical switch 25 is reflected by the mirrors 26a and 26b. Further, as shown in (c) Path 2 (P2) of FIG. 10, the light sent back to the optical switch 25 is controlled to be turned on and off again by using the piezoelectric element 25b. In this way, since the light generator controls the light on / off in the double path (P1, P2), a significantly higher on / off ratio can be obtained as compared with the single path, and the light leaks from the optical switch 25. It is possible to effectively prevent the output.

However, since high-speed on / off control is indispensable for acquiring the ringdown signal, if light is passed through an arbitrary position when using a double path, a delay in switching time occurs. Therefore, by passing light at a position at the same distance from the surface of the optical crystal 25a to which the piezoelectric element 25b is attached (P1, P2), both a high on / off ratio and high-speed on / off control can be achieved at the same time.

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

Although the carbon isotope analyzer according to the first aspect has been described above, the carbon isotope analyzer can be modified in various ways without being limited to the above-described embodiment. Hereinafter, another aspect of the carbon isotope analyzer will be described with a focus on changes from the first aspect.

[Second aspect of carbon isotope analyzer for plant sample analysis]
Conventionally, quantum cascade lasers (hereinafter also referred to as "QCL") have fluctuations in oscillation wavelengths, and since the absorption wavelengths of ¹⁴ C and ¹³ C are adjacent to each other, carbon isotope analysis such as that used for ^{14 C analysis is performed.} It was considered difficult to use as a light source for the device. Therefore, the present inventors have completed a compact and easy-to-use carbon isotope analyzer by independently developing an optical comb light source that generates an optical comb from one light source (see Patent Document 2).
Then, the present inventors have completed a light generator that generates high-power (high-intensity) light with a narrow line width as described above in order to further improve the analysis accuracy of the carbon isotope analyzer. As a result of studying further applications of the light generator, the present inventors have determined fluctuations in the oscillation wavelength of the light emitted from the QCL by the beat signal measuring device using the narrow line width light generated from the above-mentioned light generator as a frequency reference. I came up with the idea of correcting it. As a result of conducting research based on this idea, we have completed a compact, easy-to-use, and highly reliable light generator that uses a light source other than an optical frequency comb as the main light source, and a carbon isotope analyzer that uses it.

FIG. 14 is a diagram showing an outline of the carbon isotope analyzer 1B according to the second aspect. The carbon isotope analyzer 1B of FIG. 14 is the same as the light generator 20A of FIG. 1 except that the light generator 20 spectroscope 10 of FIG. 1 is replaced with the light generator 20B and the spectroscope 10B of FIG. It has the configuration of.
The light generator 20D includes a light source 23, an optical switch 25 that controls on / off of light from the light source 23, and mirrors 26a and 26b that reflect the light from the light switch 25 and send the light back to the light switch 25. The optical path 21 is not particularly limited, but for example, an optical fiber can be arranged.
The light generator 20 further includes mirrors 26c, 26d, 26e that introduce the light from the optical switch 25 into the optical spectroscope 10A.
As the light source 23, various light sources can be used without particular limitation. Details will be described later.
Various optical switches 25 can be used without particular limitation, but it is preferable to use an acousto-optic modulator (hereinafter, also referred to as “AOM”) including an optical crystal 25a and a piezoelectric element 25b. ..
The light generator 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 having a narrow line width in which one light frequency region is 4500 nm to 4800 nm, and a light from the main light source 23 and an optical comb source 28a. A light detector 28b for measuring a beat signal generated by a frequency difference of light from the light source is provided. As the optical comb source 28a, the light source according to the first embodiment described above can be used.
A part of the light from the main light source 23 is sent to the light detector 28b via the branching means 29a arranged on the optical fiber 21 and the branching means 29b arranged on the optical axis of the light from the optical comb source 28a. By sending the light, a beat signal can be generated by the frequency difference between the light from the main light source 23 and the light from the optical comb source 28a.
The carbon isotope analyzer 1B provided with the light generator 20B is free to design and maintain the carbon isotope analyzer 1B because the main light source is not limited to the optical comb and a general-purpose light source such as QCL can be used. The degree is high.
As the light generator 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 generator that simply generates light in the 4.5 μm band, which is the absorption wavelength of the radioactive carbon dioxide isotope ¹⁴ CO _{2, and has a compact device size, will be described as an example.}

<Cooling and dehumidifying device>
As shown in FIG. 14, the spectroscopic device 1a may further include a Peltier element 19 for cooling the optical resonator 11 and a vacuum device 18 for accommodating the optical resonator 11. ^Since the light absorption of 14 CO ₂ is temperature-dependent, by lowering the set temperature in the optical resonator 11 with the Perche element 19, the absorption line of ¹⁴ CO _{2 and} ^{the absorption line of 13} CO ₂ and ¹² CO ₂ can be obtained. This is because it becomes easier to distinguish between the _two , and the absorption intensity of ¹⁴ CO 2 becomes stronger. Further, by arranging the optical resonator 11 in the vacuum apparatus 18, it is possible to prevent the optical resonator 11 from being exposed to the outside air and reduce the influence of the external temperature, thereby improving the analysis accuracy.
As the 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, or the like can be used. The Peltier element 19 is preferably used from the viewpoint of miniaturizing the spectroscopic device 10, and a liquid nitrogen water tank or a dry ice tank is preferably used from the viewpoint of reducing the manufacturing cost of the device.
The vacuum device 18 is not particularly limited as long as it can accommodate the optical resonator 11, irradiate the light from the light generator 20 into the optical resonator 11, and transmit the transmitted light to the light detector. Various vacuum devices can be used.
A dehumidifying device may be provided. At that time, dehumidification may be performed by a cooling means such as a Peltier element, or dehumidification may be performed by a membrane separation method using a polymer film for removing water vapor such as a fluorine-based ion exchange resin film.

When using a carbon isotope analyzer 1 described above in microdosing, detection sensitivity to radiocarbon ^{14 C} is about "0.1dpm / ml" is assumed. In order to achieve this detection sensitivity of "0.1 dpm / ml", it is not enough to 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 administered radioactivity is not as large as that of humans, so both the lower limit of detection radioactivity and the abundance sensitivity can be solved by administering the dose so as to exceed the lower limit of the analyzer. Therefore, the abundance sensitivity (analyzable isotope ratio) is ^{about 10-12} (natural isotope ratio) at the lower limit.
In plant sample analysis, it is preferable that the wavelength of the absorption line does not deviate and the line width is narrow, even if higher sensitivity is not required as compared with the biological sample. In this respect, the carbon isotope analyzer 1 can solve this problem by using a stable light source using "optical frequency comb light" for CRDS. According to the carbon isotope analyzer 1, an advantageous effect is exhibited that the measurement can be performed even on a sample containing a low concentration of radiocarbon isotopes.
The prior literature (Kazuo Hiromoto et al., "Design study of 14C continuous monitoring based on cavity ring-down spectroscopy", Proceedings of the Spring Annual Meeting of the Atomic Energy Society of Japan, March 19, 2010, P432) is related to nuclear power generation. In connection with the concentration monitoring of spent fuel in Japan, it is disclosed that ^{CRDS measures the concentration of 14 C in carbon dioxide.} However, the signal processing method using the fast Fourier transform (FFT) described in the prior art achieves a detection sensitivity of "0.1 dpm / ml" because the baseline fluctuation becomes large although the data processing becomes faster. It's difficult to do.

FIG. 15 (quoted from Applied Physics Vol.24, pp.381-386, 1981) shows the absorption wavelengths of ^{analytical samples 12} C ¹⁶ O ₂ , ¹³ C ¹⁸ O ₂ , ¹³ C ¹⁶ O ₂ , and ¹⁴ C ¹⁶ O _2. The relationship of absorption strength 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 due to the pressure and temperature of the sample. Therefore, it is preferable that the pressure of the sample is atmospheric pressure or less and the temperature is 273K (0 ° C.) or less.

As described above, since ^{the absorption intensity of 14} CO ₂ is temperature-dependent, it is preferable to set the set temperature in the optical resonator 11 as low as possible. The specific set temperature in the optical resonator 11 is preferably 273 K (0 ° C.) or less. The lower limit is not particularly limited, but from the viewpoint of cooling effect and economy, it is preferable to cool to 173K to 253K (-100 ° C to -20 ° C), particularly to about 233K (-40 ° C).
The spectroscope may further include vibration absorbing means. This is because it is possible to prevent the mirror spacing from shifting due to vibration from the outside of the spectroscope and improve the 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, a device capable of applying vibration of the opposite phase of the external vibration to the spectroscopic device can be used.

[First aspect of carbon isotope analysis method]
Radioisotope ^14C will be described as an example for analysis.

(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 source of the ^{radioactive isotope 14 C.} Then, after the biological activity of the plant sample is stopped, each part of the plant sample is cut (sampled). After that, it is preferable to dry the plant sample. Further, the plant sample may be cut into small pieces.

(B) A plant sample is heated and burned to generate a gas containing carbon dioxide isotope ¹⁴ CO ₂ ^{from a radioactive isotope 14 C source.} In this case, for example, a plant sample photosynthesized in an environment containing ¹⁴ CO ₂ gas can be used by using the system described later. _{N 2} O and CO are removed from the obtained gas.

(C) It is preferable to remove water from the ^{obtained 14} CO _2. For example at the carbon dioxide isotope generating apparatus 40, the ¹⁴ CO ₂ or passed over drying agent such as calcium carbonate, it is preferred to remove water by condensation of moisture by cooling the ¹⁴ CO _2. ^{14 This} is because the decrease in mirror reflectance due to icing and frosting of the optical resonator 11 due to the moisture contained in CO _{2 reduces the detection sensitivity, and thus the analysis accuracy is improved by removing the moisture.} Considering the spectroscopic process, it is preferable to cool ^{the 14} CO ₂ before introducing the ¹⁴ CO _{2 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 in the optical resonator 11 having a pair of mirrors 12a and 12b as shown in FIG. Then, ^{it is preferable to cool 14} CO ₂ to 273 K (0 ° C.) or less. This is because the absorption intensity of the irradiation light is increased. Further, it is preferable to keep the optical resonator 11 in a vacuum atmosphere. This is because the measurement accuracy is improved by reducing the influence of the 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. Further, the first light is transmitted to the second optical fiber 22 that branches from the first optical fiber 21 and merges at the confluence on the downstream side of the first optical fiber 21, and the second optical fiber 22 transmits the second light having a wavelength longer than that of the first light. To generate. The intensities of the obtained first light and the second light may be amplified by using amplifiers (not shown) having different bands.
Then, light in the 1.3 μm to 1.7 μm band is generated from the first optical fiber 21 on the short wavelength side, and light in the 1.8 μm to 2.4 μm band is generated from the second optical fiber 22 on the long wavelength side. Next, the second light is merged 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 the absorption wavelength of the ^{carbon dioxide isotope 14} CO _{2 is changed from the difference in frequency.} As the light in the 5 μm band, an optical comb having a wavelength in the mid-infrared region of 4.5 μm to 4.8 μm is generated as the irradiation light. At that time, high-intensity light can be generated by using a long-axis crystal having a length in the longitudinal direction longer than 11 mm as the nonlinear optical crystal 24.

(F) The carbon dioxide isotope ¹⁴ CO ₂ is irradiated with irradiation light to resonate. At that time, in order to improve the measurement accuracy, it is preferable to absorb the vibration from the outside of the optical resonator 11 so that the intervals between the mirrors 12a and 12b do not deviate. Further, it is preferable to irradiate the first optical fiber 21 with the other end on the downstream side in contact with the mirror 12a so that the irradiation light does not come into contact with air. Then, the intensity of the transmitted light from the optical resonator 11 is measured. The transmitted light may be separated and the intensity of each separated transmitted light may be measured.

(G) Calculate the ^{carbon isotope 14} C 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 embodiment, and various modifications can be made. Hereinafter, another aspect of the carbon isotope analysis method will be described focusing on the changes from the first aspect.

[Second aspect of carbon isotope analysis method]
The second aspect of the carbon isotope analysis method replaces the above-mentioned step (e) with the following step.
(A) The carbon isotope analysis method generates an optical comb composed of a bundle of light having a narrow line width in which the frequency domain of one light is 4500 nm to 4800 nm.
(B) Next, as shown in FIG. 16A, the spectrum of the light of one of the optical combs is displayed at the center of the absorption wavelength region of the test object in the optical spectrum diagram of the intensity with respect to the frequency.
(C) The light from the optical comb is transmitted to the optical fiber for measuring the beat signal.
(D) The object to be inspected is irradiated with light from a light source, and the amount of light absorbed is measured by an optical resonator (CRDS).
(E) A part of the light from the light source is branched into the optical fiber for measuring the beat signal, and the beat signal is generated by the frequency difference between the light from the light source and the light from the optical comb source. At that time, as shown by the arrow in FIG. 16B, a beat signal may be generated while scanning a wide range of frequencies such as (1), (2), and so on. Further, as shown in FIG. 16C, a beat signal may be generated in a desired frequency region.
The wavelength of the light radiated to the test object obtained from the beat signal obtained in the step (e) is recorded together with the amount of light absorbed in the steps (f) and (d). Based on those records, the exact amount of light absorption of the test object is measured.
In the present invention, although the phase lock by the optical comb is not intentionally performed, accurate measurement can be realized by a simple measurement system.

^[14 CO ₂ labeling system for plant samples]
^{The 14} CO ₂ photosynthetic labeling system for plant samples will be described below. In order to carry out an experiment to evaluate the dynamics of the photosynthetic product produced in the above-ground part of rice to move to the root, it is desirable to introduce ^{a plant sample into a 14} CO ₂ ^{environment and label it with 14 C by photosynthesis.} Therefore, previous studies (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, 4 , 743-753 (2016)) ^{, using the chemical reaction of 14} C-labeled sodium hydrogen carbonate solution (NaH ₁₄ CO ₃ , 37 MBq / mL, PerkinElmer) and weak acid (lactic acid, CH ₃ CH (OH) COOH). We built a labeling system. An outline is shown in FIG.
FIG. 17 is a conceptual diagram of the ¹⁴ CO _{2 photosynthetic labeling system 80 for plant samples.} ^{The 14} CO ₂ photosynthetic labeling system 80 for plant samples ^{includes a vial 81 for generating 14} CO ₂ 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. .. Further, a pipe 88a for connecting each member in series and a pipe 88b for connecting between the circulation pump 86 and the sample bag 83 are further provided. The pipe 88a is provided with a valve 89a between the vial 81 and the sample bag 83, a valve 89b between the sample bag 83 and the desiccator 85, and the pipe 88b is provided with the valve 89c. By opening and closing each of the valves 89a to 89c and operating the circulation pump 86, gas can be circulated between the sample bag 83 and the desiccator 85. A light source 82 is arranged above the desiccator 85. The desiccator 85 can be made of acrylic.
The volume of the sample bag 83 is preferably 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 gas in the sample bag 83 can be easily introduced into the desiccator 85.

Next, a ^{method of generating 14} CO ₂ gas will be described. ^{When lactic acid is added dropwise to the 14} C-labeled sodium hydrogen carbonate solution in the vial 81, ¹⁴ CO ₂ gas is generated by the following chemical reaction.
NaH ₁₄ CO ₃ + CH ₃ CH (OH) COOH
→ ¹⁴ CO ₂ + H ₂ O + CH ₃ CH (OH) COONa
At this time, if from sufficient Okere amount of amount of sodium bicarbonate lactate, ¹⁴ C in the vial 81, all become ¹⁴ CO ₂ gas, ¹⁴ CO ₂ gas generated in ¹⁴ C-bicarbonate present in the vial 81 It can be easily calculated from the amount of sodium solution.

The operation of the above-mentioned system will be described. (A) First, the valves 89a to 89c are opened and closed so that only the vial 81 and the 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 the ¹⁴ CO _{2 gas.} (C) After that, the gas in the system is circulated using the circulation pump 86. As a result, the plant sample inside the desiccator 85 is taken into the body of ¹⁴ CO _{2 by photosynthesis.} (D) It is preferable that the light source 82 (here, the LED light) installed above the desiccator 82 promotes photosynthesis of the plant sample inside the desiccator 85.

[Plant sample analysis system]
The present invention also relates to a plant sample analysis system including the above-mentioned carbon isotope analyzer for plant sample analysis and a ¹⁴ CO ₂ photosynthetic labeling system (unit) for plant samples. According to such a system, plant sample analysis can be performed efficiently. It is preferable that each member is in the same premises, but the present invention is not limited to this, and each member may be arranged in a different premises.

(Other embodiments)
As mentioned above, the invention has been described by embodiment, but the statements and drawings that form part of this disclosure should not be understood to limit the invention. Various alternative embodiments, examples 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 radioisotope ^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 the ¹² C and ¹³ C analysis is ^{performed as the absorption line analysis of 12} CO ₂ and ¹³ CO ₂ , it is preferable to use the light in the 2 μm band or the 1.6 μm band.
^When performing absorption line analysis of 12 CO ₂ and ¹³ CO ₂ , the mirror spacing is preferably 10 to 60 cm, and the radius of curvature of the mirror is preferably the same as or greater than the mirror spacing.
Although ¹² C, ¹³ C, and ¹⁴ C each behave chemically the same, the natural abundance ratio of the ^{radioactive isotope 14 C is lower than that of the stable isotope elements 12} C and ¹³ C, so that the radioactive isotope is a radioactive isotope. ¹⁴ C can be observed in various reaction processes by increasing its concentration by artificial manipulation and performing accurate measurement.
The light generator (optical switch) described in the first embodiment can be used for various purposes because it can control the on / off of light with high accuracy. For example, a measuring device, a medical diagnostic device, an environmental measuring device (dating device), and the like including a part of the configuration described in the first embodiment can be manufactured.
The optical frequency comb described in the first embodiment is a light source in which the longitudinal mode of the laser spectrum is arranged at equal frequency intervals with extremely high accuracy, and is a new light source with high functionality in the fields of precision spectroscopy and high-precision distance measurement. Expected. In addition, since there are many absorption spectra of substances in the mid-infrared region, it is important to develop an optical frequency comb light source 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.
As described above, it goes without saying that the present invention includes various embodiments not described here. Therefore, the technical scope of the present invention is defined only by the matters specifying the invention relating to the reasonable claims from the above description.

[ ^{Measurement of 13} C-labeled corn sample]
A basic experiment was conducted using a ^{13 C-labeled corn sample.} The sample was a 13C-labeled foliage / root system sample of corn for a purpose different from this study, and was transferred by Dr. Mana Nakata, Graduate School of Bioagricultural Sciences, Nagoya University. The sample was finely cut and pulverized from the beginning. A total of six samples of 3 samples each with or without ^{labels, 13} C-labeled There the sample No.1~3 (1,2: foliage, 3: root ^system), Nanba4～ samples without ¹³ C-labeled It was labeled as 6 (4,5: foliage, 6: root system).
In addition, an acetanilide (C ₈ H ₉ NO) sample (solid) was measured as a reference sample for the natural isotope ratio. This sample had been analyzed for ¹³ C isotope ratio by Isotope Ratio Mass Spectrometry (IRMS) prior to this study. Approximately 2 mg of each sample was weighed, placed in an alumina sample boat, and then inserted into a solid sample combustor. First, _{in order to confirm that the CO 2} ^{absorption spectrum derived from the plant sample can be obtained by the 14} C-CRDS analysis system, the unlabeled sample (stem and leaf part) of No. 5 was introduced, and carbon dioxide containing ¹² C and ^{13 C was introduced.} A relatively wide range of absorption spectra was measured by scanning ^{2209.25-2210.0 cm -1} , a wave region in which relatively large absorption of carbon is present. The temperature of the gas cell was maintained at 283 K and the pressure at the time of measurement was 20 mbar. ^The spectrum obtained for the 13 C-labeled corn foliage is shown in FIG.
Absorption peaks of N ₂ O, CO, and H ₂ O were confirmed, but clear absorption peaks of ¹³ CO ₂ and ¹² CO _{2 were obtained without being buried in them.} From this, it was shown that _{the CO 2} absorption spectrum derived from the plant sample can be measured. Hereinafter, the scan range was set to 2209.75 to 2210.0 cm ^-1 , in which ^{both peaks of 13} CO ₂ and ¹² CO _{2 are 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 maintained at 283 K and the pressure at the time of measurement was 20 mbar. Examples of the acquired spectra (No. 1 and No. 5, both foliage) are shown in FIGS. 19A and 19B, respectively. Here, the vertical axis in the figure is a linear scale instead of a logarithm in order to clarify the change in peak intensity. Since there are individual differences in the carbon content in the sample due to the slight difference in the sample introduction amount, FIGS. 20A and 20B are standardized with an intensity of ¹² CO _{2 on the vertical axis from FIGS. 19A and 19B.}

From this, it was shown that the ¹³ C-labeled sample No. 1 clearly had a higher ^{relative intensity of 13} CO _{2 absorption than the unlabeled sample No. 5.} In addition, the evaluated isotope ratio was about twice as large as that of No. 5 while that of No. 5 was the natural isotope ratio. All samples were measured 3 times (n3), and the evaluated isotope ratios are shown in Table 1 together with the evaluation results by IRMS. Note in the table, D13C the reference sample of the natural ¹³ C isotope ratio (Pee Dee Belemnite Ore: PDB).. Value of ^{(13 C / 12 C 0.012021%} ) (J Meija, et al, "Isotopic compositions of The value is based on the elements 2013 (IUPAC Technical Report) ”, Pure Appl. Chem., 88, 3, 293-306 (2016)) and is expressed by the following formula. The unit is parts per mille (per mille, ‰).
Based on the results shown in Table 1, the results of plotting the d13C measured for each sample are shown in FIG. At this time, the vertical axis was corrected according to the result of acetanilide. ^{14 The} evaluation by the C-CRDS analysis system was in good agreement with the evaluation by IRMS. The absorption line of ¹³ CO ₂・¹² CO ₂ existing in the wave number region (2208 cm ^{-1 to} 2212 cm ^{-1 ) measurable 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, it was shown that the isotope ratio of stable carbon in plants can be estimated by this method.

^[14 CO ₂ labeling system for plant samples]
^{In order to confirm the 14} C labeling of plant samples, an experiment was conducted in which rice samples grown under the same conditions were ^{labeled by a 14} CO _{2 photosynthetic labeling system.} ^Using 20 μL (= 740 kBq) of ^{14 C-sodium hydrogen carbonate solution, 14} CO ₂ gas was generated and the rice sample was ^{exposed (labeled) to 14} CO ₂ gas for 50 minutes. For comparison of results, one individual was not labeled. After labeling, it was divided into foliage and root system, and after terminating biological activity by immersing it in liquid nitrogen, it was dried in a dryer. The sample was cut into small pieces and the absorption spectrum was obtained by a ^{14 C-CRDS analysis system.
The results of the 14} C-labeled and unlabeled foliage samples are shown in FIGS. 22A and 22B, respectively. The results showed that the labeled sample had a distinct peak of ¹⁴ CO ₂ ^{not found in the unlabeled sample, indicating that this system allows 14} CO ₂ labeling by photosynthesis.

[ ¹⁴ C Demonstration of carbon dynamics evaluation of rice by C tracer analysis]
It was confirmed that plant sample analysis was possible by the above-mentioned ^{14 C-CRDS analysis system, a 14} C labeling system by photosynthesis was constructed, and its operation was confirmed. ^{Therefore, an experiment was conducted in which 14} CO ₂ was absorbed by a rice sample by photosynthesis and the dynamics of the photosynthetic product moving to the roots were evaluated. As rice samples, two groups (20 individuals each, 40 individuals in total) of rice (variety: Nihonbare) cultivated in an artificial meteorological machine for 10 days under hydroponic conditions were prepared. One was grown normally in an artificial meteorological machine (Control), and the other was mixed with polyethylene glycol (PEG) in a hydroponic solution to generate pseudo-dry stress. PEG can absorb a large amount of water inside, and can suppress the absorption of water into plants. All the individuals in the Control group and the PEG group were introduced into the desiccator of the labeling system shown in FIG. 17 described above.

Since the number of individuals is 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 sample was taken out from the desiccator, and 5 individuals each of Control and PEG were grouped, and after a certain period of time (immediately after, 30 minutes, 60 minutes, 120 minutes), the foliage (Shoot) and the root system (Root) were formed. After separating, each was immersed in liquid nitrogen to stop biological activity. This work required about 5 minutes, and the median of the results was the time when biological activity was stopped.
Then, it was dried with a drier, the weight of each sample was weighed for each individual, and it was cut and pulverized. Such work is called sampling. ^The amount of 14 C was measured at 1.0 mg for the foliage, which is expected to be relatively large, and 1.5 mg for the root system, and the absorption spectrum was obtained by the ^{14 C-CRDS analysis system.} The gas cell was maintained at 283 K, and the pressure at the time of measurement was about 15 mbar in the root system and about 50 mbar in the foliage. In the case of foliage, the pressure is high because the ^{amount of 14C} is high, 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 becomes ^{independent of the amount of 14} C, and the peak zenith 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 an example of the acquired absorption spectrum, the results of the root system and foliage of the same individual sampled immediately after the PEG group are shown in FIGS. 23A and 23B, respectively.

The foliage and root system of 5 individuals in each group of each group were measured, and the ¹⁴ C isotope ratio was evaluated. The horizontal axis represents the elapsed time from the label and the vertical axis represents the ¹⁴ C isotope ratio, and the results are divided into the root system and the foliage and plotted in FIGS. 24A and 24B, respectively. In addition, FIG. 26 shows a diagram in which the ratio of isotope ratios of the root system and the foliage is taken and the horizontal axis is similarly the elapsed time from the label. Comparing the isotope ratios evaluated in the foliage and root system samples, it was found that the root system was several orders of magnitude lower and the difference became smaller as the elapsed time increased (the ratio of root to foliage increased). .. ¹⁴ C absorbed by photosynthesis in the foliage is valid as a result of migration to the root system. ^{Further, the 14} C isotope ratio can be converted into the radioactivity amount A (unit: Bq) in the sample by the following formula.
Here, NA is the Avogadro constant (= 6.022 × 10 ²³ ), and λ is the ¹⁴ C decay constant. ^{The horizontal axis shows the elapsed time from the label, and the vertical axis shows the 14} C time change in the root system and the foliage as the amount of radioactivity in the sample, which are shown in FIGS. 25A and 25B. The distribution rate to the root system, Droot, is the ratio of the sum of the radioactivity of the foliage and the root system (total radioactivity) to the radioactivity of the root system, and is calculated by the following formula.
Here, Aroot and Ashoot are the amount of radioactivity evaluated in the root system and foliage, respectively. The distribution rate for each elapsed time is shown in FIG. From the results, PEG (with drought stress) had a low radioactivity value as a whole. This indicates that drought stress reduces the functions related to foliage growth and photosynthesis. In fact, the size and weight of the foliage of the PEG group sample are relatively small, and the ^{amount of 14} CO ₂ taken up as shown in FIGS. 24A and 24B and 25A and 25B is relatively small. On the other hand, ^{the slopes of the 14} C isotope ratio, the amount of radioactivity, and the distribution rate to the roots of the root system were larger in PEG than in Control. This indicates that photosynthetic products are more rooted to adapt to drought stress. On the other hand, the results of PEG showed ^{a tendency of saturation in the 14} C isotope ratio, the amount of radioactivity, and the distribution rate to the roots of the root system. The roots not only receive photosynthetic products from the foliage, but also excrete them as _{CO 2 by respiration.} It is known that root respiration rate increases under drought stress (Nakada (Kano) Mana et al., "Aeration tissue formation and root respiration characteristics involved in soil dry stress response of rice root system", 243rd Japan See Lecture by the Crop Science Society of Japan, p.192 (2017)), ^{suggesting that the 14} C supply rate ^{from the foliage to the root and the 14} C excretion rate by respiration may have reached an equilibrium state. CRDS is a method for analyzing gas, and in the future, in addition to the same evaluation as in this experiment, it is expected to develop a new evaluation method such as evaluating the amount ^{of 14} CO _{2 in the exhaled breath from the root system.} To.

From the above, it is possible to acquire the temporal trend of the photosynthetic products produced by the leaves being transported to the roots and evaluate the difference between the presence and absence of drought stress, and demonstrate the dynamic evaluation of the photosynthetic products in rice by the ^{14 C-CRDS analysis system.} did. Than currently obtained results and evaluated sensitivity ^{(14 C / 12 C~2.0 × 10} -11), even if the orders of magnitude smaller dosage amount of radioactivity can similar measurement, for the introduction sample, less It turned out that it is possible to evaluate by quantity.

Focusing on the application ^{of the 14} C tracer in plant physiology as the application destination of the ¹⁴ C analysis system based on the mid-infrared CRDS ^{(14 C-CRDS analysis system), through a demonstration experiment in which rice samples are labeled with 14} C and their dynamics are evaluated. , There is usefulness in plant physiology of this method. After constructing an introduction part for plant sample measurement and confirming its operation, it was shown that it is possible to measure the carbon isotope ratio in the plant sample by a basic experiment using a ^{13C-labeled corn sample.} Further, to build a ¹⁴ CO ₂ labeling system for plant samples, the rice samples grown in the presence / absence condition of drought stress ^{14 C-labeled,} time ^{to 14 C} taken from leaves by photosynthesis is transported to the roots I got a trend. ^The carbon dynamics of 14 C moving from leaves to roots could be clearly evaluated, and it was clarified that the dynamics differ depending on the presence or absence of drought stress. At the same time, since it has several orders of magnitude higher sensitivity than existing analytical methods, it is expected that the same evaluation can be performed with a smaller dose of radioactivity and sample.
As future prospects, ^{in addition to the development of 14} CO ₂ measurement method in exhaled roots, measurement of foliage exudate, dynamic evaluation of leaves and stems, taproot and lateral roots, and ¹⁴ C analysis in metabolites. It is expected that this method will be applied to various analyzes that could not be realized because there was no suitable analysis method so far.

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 Perche element 20A, 20B optical generator 21 1st optical fiber 22 2nd optical fiber 23 light source 24 non-linear optical crystal 25 optical switch 26a~26e mirror 28 beat signal measuring instrument 29 optical branching device 30 calculating unit 40 the carbon dioxide isotope generating apparatus 80 for plant samples ¹⁴ CO ₂ photosynthetic labeling system 81 vial 83 sample bag 85 desiccator 86 Circulation pump 88a, 88b Piping 86 Circulation pump 89a-89c Valve

Claims (16)

A carbon dioxide isotope generator equipped with a combustion part that generates a gas containing carbon dioxide isotopes from carbon isotopes,
Light generator and
A carbon isotope analyzer for plant sample analysis, comprising an optical resonator having a pair of mirrors, a spectroscopic apparatus including a photodetector for detecting the intensity of transmitted light from the optical resonator. The carbon isotope analyzer for plant sample analysis according to claim 1 ^{, wherein the carbon isotope is a radiocarbon isotope 14} C, and the carbon dioxide isotope is a radiocarbon dioxide isotope ¹⁴ CO _2. .. The plant sample analysis according to claim 1 or 2, wherein the combustion unit includes a combustion furnace arranged substantially perpendicular to the direction of gravity, a sample boat, a carrier gas supply tank, and a dehumidifying device. Carbon isotope analyzer for. The invention according to any one of claims 1 to 3, further comprising a carbon dioxide isotope trap provided between the carbon dioxide isotope generator and the spectroscopic device, which comprises a cooling device for freezing the carbon dioxide isotope. The carbon isotope analyzer for plant sample analysis described. The light generator includes a light source, an optical switch for controlling the 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 carbon isotope analyzer for plant sample analysis according to any one of the above items. The carbon isotope analyzer for carbon dynamics analysis in a plant material according to claim 5, wherein the optical switch is an acousto-optic modulator. The light generator includes a branching means for branching light from the light source, a condensing lens for condensing light from the branching means, and the condensing lens and the branching means for reflecting light from the condensing lens. The carbon isotope analyzer for plant sample analysis according to any one of claims 1 to 6, further comprising a mirror that sends light back to the light source via the light source. The carbon isotope analyzer for plant sample analysis according to any one of claims 1 to 7, wherein the light source generates optical frequency comb light. The carbon isotope analyzer for plant sample analysis according to any one of claims 1 to 8, wherein the light source is a fiber laser. The carbon isotope analyzer for plant sample analysis according to any one of claims 1 to 7, 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 line width in which the frequency region of one light is 4500 nm to 4800 nm, a beat generated 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 equipped with an optical detector for measuring a signal,
The carbon isotope analyzer for plant sample analysis according to any one of claims 1 to 7. The carbon isotope analyzer for plant sample analysis according to any one of claims 1 to 11.
^A system for plant sample analysis including a vial for generating 14 CO ₂ gas, a sample bag for accumulating the generated gas, a desiccator into which a plant sample is introduced, and a ¹⁴ CO _{2 photosynthetic labeling unit for a plant sample provided with a circulation pump.} ¹⁴ Vials that generate CO _{2 gas and}
A sample bag that accumulates the generated gas and
The desiccator into which the plant sample is introduced and
^{A 14} CO ₂ photosynthetic labeling system for plant samples with a circulation pump. ^{14 The} process of photosynthesizing plant samples in an environment containing CO _{2 gas,}
A step of producing a carbon dioxide isotope from the carbon isotope in the plant sample, a step of filling the optical resonator with the carbon dioxide isotope, and a step of filling the optical resonator.
A step of irradiating the optical resonator with irradiation light having an absorption wavelength for the carbon dioxide isotope, and
A step of measuring the intensity of transmitted light obtained when the carbon dioxide isotope is irradiated with the irradiation light and resonated, and
A carbon isotope analysis method for plant sample analysis, which comprises a step of calculating a carbon isotope concentration from the intensity of transmitted light. Between the step of irradiating the irradiation light and the step 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 claim 14, further comprising a step of controlling the above. The 14th or 15th claim, wherein as the irradiation light, an optical comb having a wavelength in the mid-infrared region 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.