Specimen measurement device and specimen measurement method

Abstract

To provide a specimen measurement device and a specimen measurement method with which it is possible to simultaneously measure a plurality of indices related to the physiological activity of a plant and grasp the growth state of the plant in real time.SOLUTION: A specimen S which is the object of inspection is fixed in place by a holder unit 20 while being immersed in a culture fluid inside of a specimen accommodation unit 10. A laser beam emitted from a laser oscillation unit 30 is reflected by a first mirror unit 40 and further concentrated by a condenser lens 50 before passing through the specimen accommodation unit 10. The degree of deviation of the reflected light from the optical axis at this time is detected by a polarization degree detection unit 60, which makes it possible to grasp the dynamic process of a physiologically active substance on the surface of the specimen S. Furthermore, the intensities of fluorescence radiated by excitation of fluorescent molecules deriving from the specimen S and fluorescent probe molecules in the culture fluid are respectively detected by a fluorescence detection unit 80.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a specimen measurement device and a specimen measurement method, and more particularly to a specimen measurement device and a specimen measurement method that can simultaneously measure multiple indices related to the physiological activity of biological organs, including plants, and grasp the biological state in real time. [Background technology]

[0002] In the past, the management of plant growth conditions at plant cultivation sites involved visual observation of changes in the color of the leaves and stems, the presence or absence of surface spots, or changes in the time it takes for new leaves and buds to appear, and the growth conditions were determined based on experience and intuition, or by changes in the growth of the plants, stem thickness, leaf size, or yield.

[0003] On the other hand, in recent years, large-scale plant cultivation has become mainstream, and the area under cultivation is also increasing. As the cultivation area increases, it becomes difficult for producers and others to manage the growth status of the entire plant group under cultivation using the same visual inspection method as in the past.

[0004] Therefore, technologies have been proposed that diagnose the growth state of plants from their physiological functions without visually observing their health state. For example, there is a technology that manages the health state of plants by evaluating the photosynthetic function, which is closely related to the health state of plants (Patent Document 1).

[0005] The technology described in Patent Document 1 is a technology that evaluates the photosynthetic function of plants by driving the photochemical reaction of photosynthesis with light energy absorbed by chlorophyll and measuring the chlorophyll fluorescence emitted by chlorophyll at this time.Since chlorophyll fluorescence is measured as an image, it is called the chlorophyll fluorescence image measurement method.

[0006] This chlorophyll fluorescence imaging method measures the chlorophyll fluorescence emitted by chlorophyll, which is important for photosynthesis, and therefore allows for direct diagnosis of photosynthetic reactions. This makes it possible to diagnose physiological dysfunction in plants, in other words, disorders that cannot be seen with the naked eye. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2001-299090 Summary of the Invention [Problem to be solved by the invention]

[0008] However, the measurement method disclosed in the aforementioned Patent Document 1 measures only chlorophyll fluorescence intensity as an index related to the physiological activity of plants. Therefore, when measuring other indexes, such as the inflow and outflow of physiologically active substances near the plant surface, the dissolved oxygen concentration, and the quantum yield of photosystem II, it is necessary to prepare separate measuring devices for each index, which requires a long time to measure all the indexes and has the problem that each index cannot be obtained simultaneously in real time.

[0009] The present invention was devised in consideration of the above points, and relates to a specimen measurement device and a specimen measurement method that can simultaneously measure multiple indicators related to the physiological activity of biological organs, including plants, and grasp the biological condition in real time. [Means for solving the problem]

[0010] In order to achieve the above-mentioned object, the specimen measurement device of the present invention comprises a specimen storage section capable of storing a specimen to be tested, a holder section for holding the specimen, a laser oscillator section for emitting laser light in a first direction, a first mirror section for reflecting the laser light emitted from the laser oscillator section as reflected light in a second direction, a focusing lens section provided on the optical axis of the reflected light and focusing the reflected light toward the vicinity of the specimen, a polarization detection section for detecting the polarization, which is the amount of deviation of the reflected light from the optical axis that has passed through the specimen storage section, a second mirror section for transmitting the first fluorescence in a third direction and reflecting the second fluorescence in a fourth direction, a first fluorescence detection section for detecting the fluorescence intensity of the first fluorescence that has passed through the second mirror section, and a second fluorescence detection section for detecting the fluorescence intensity of the second fluorescence reflected by the second mirror section.

[0011] Here, by providing a specimen storage section capable of storing the specimen to be tested and a holder section for holding the specimen, the specimen can be placed and held in the specimen storage section, preventing the specimen from moving during measurement. Therefore, the laser light described below can be passed near the plant specimen, thereby improving measurement sensitivity and measurement accuracy.

[0012] Furthermore, by providing a laser oscillator that emits laser light in a first direction, the laser light passes through the vicinity of the specimen, allowing multiple indices related to the specimen (for example, indices related to physiological activity if the specimen is a plant) to be measured simultaneously.

[0013] Furthermore, by providing a first mirror portion that reflects the laser light emitted from the laser oscillation portion in a second direction as reflected light, the laser light emitted from the laser oscillation portion can be adjusted to be directed toward the target transmission path.

[0014] Furthermore, by providing a focusing lens unit that is located on the optical axis of the reflected light and focuses the reflected light toward the vicinity of the specimen, it is possible to focus almost all of the reflected light reflected by the first mirror unit near the measurement site of the specimen, thereby enabling measurement of each site of the specimen with high accuracy and sensitivity.

[0015] Furthermore, by providing a polarization detection unit that detects the degree of polarization, which is the amount of deviation from the optical axis of the reflected light that has passed through the specimen storage unit, it is possible to obtain information on dynamic processes, such as substance transport and biochemical reactions, on the specimen surface during the specimen's physiological activity from the measurement results of changes in the refractive index gradient caused by changes in the temperature gradient and concentration gradient near the specimen through which the laser light has passed.

[0016] Furthermore, when reflected light passes through the vicinity of the specimen, the first fluorescence and the second fluorescence are emitted by excitation of fluorescent molecules such as chlorophyll contained in the specimen and fluorescent probes in the culture medium in contact with the specimen. By providing a second mirror section that transmits the first fluorescence in a third direction and reflects the second fluorescence in a fourth direction, when the specimen and / or the medium in its vicinity contains two different types of fluorescent molecules, these fluorescence can be transmitted and reflected in different directions, respectively.

[0017] Furthermore, by providing a first fluorescence detection unit that detects the fluorescence intensity of the first fluorescence that has passed through the second mirror unit, it is possible to obtain information on the fluorescence intensity of the first fluorescence that has passed through the second mirror unit.

[0018] Furthermore, by providing a second fluorescence detection unit that detects the fluorescence intensity of the second fluorescence reflected by the second mirror unit, it is possible to obtain information on the fluorescence intensity of the second fluorescence that has passed through the second mirror unit.

[0019] Furthermore, when one of the first and second fluorescence is fluorescence from a fluorescent probe, the inflow and outflow of oxygen on the surface of the specimen can be detected from the degree of fluorescence quenching of the fluorescent probe. Furthermore, when the other of the first and second fluorescence is chlorophyll fluorescence, information useful for photosynthesis of the specimen plant can be detected.

[0020] In addition, if a chlorophyll fluorescence measurement unit using pulse modulation that measures the quantum yield of photosystem II of the plant specimen is located near the specimen storage unit, information such as the quantum yield of photosystem II of the plant specimen can be obtained simultaneously.

[0021] In addition, the specimen is immersed in a culture solution containing a fluorescent probe within the specimen storage section, and if the specimen storage section is equipped with an oxygen concentration sensor that detects the dissolved oxygen concentration of the culture solution and a temperature sensor that detects the temperature of the culture solution, the respiration and photosynthesis status of the entire specimen plant can be obtained simultaneously from information on changes in the oxygen concentration and temperature in the culture solution.

[0022] In addition, the polarization detection unit is composed of a lens system and a position detection sensor unit, and when the reflected light is focused on the position detection sensor unit via the lens system, changes in the refractive index gradient caused by changes in the temperature gradient or concentration gradient near the surface of the specimen through which the laser light has passed can be detected with high accuracy and high sensitivity.

[0023] Furthermore, if there is a light source unit above the specimen storage unit that can be switched on and off, the status of the respiration process and photosynthesis process of the specimen plant can be switched by turning the light source unit on and off, and therefore indicators of physiological activity according to each status can be obtained.

[0024] Furthermore, when the holder unit includes a transparent plate having a through-hole formed from one side to the other side and a pressing unit that presses the specimen from one side to the other side with the specimen placed on one side of the transparent plate so that a portion of the specimen enters the through-hole and faces the other side in a convex manner, the portion of the specimen placed on the holder unit faces the other side in a convex manner through the through-hole, allowing the laser light to pass through and target the area below the convex surface of the specimen facing the other side. This makes it easy to obtain indicators of physiological activity on the front and back surfaces of plant leaves, where physiological activities such as respiration and photosynthesis are most active.

[0025] Furthermore, when the holder is a transparent cylindrical body that is a predetermined length shorter than the entire length of the specimen, the laser light can be transmitted to the vicinity of a portion of the specimen exposed through the holder while the specimen is placed in the holder, thereby easily obtaining indicators of physiological activity on the upper and lower surfaces of the leaves of the plant body, where physiological activities such as respiration and photosynthesis are most active.

[0026] In order to achieve the above-mentioned object, the specimen measurement method of the present invention is a method for measuring a specimen contained in a specimen holding section, and includes the steps of: emitting laser light in a first direction; reflecting the laser light as reflected light in a second direction; focusing the reflected light toward the vicinity of the specimen; detecting a degree of polarization, which is the amount of deviation of the reflected light that has passed through the specimen holding section from the optical axis; transmitting the first fluorescence in a third direction and reflecting the second fluorescence in a fourth direction, of first and second fluorescence emitted by excitation of fluorescent molecules such as chlorophyll contained in the specimen and fluorescent probes in a culture medium when the reflected light passes through the vicinity of the specimen; detecting the fluorescence intensity of the first fluorescence that has passed through the third direction; and detecting the fluorescence intensity of the second fluorescence that has reflected in the fourth direction.

[0027] Here, by providing a step of emitting laser light in a first direction, it is possible to simultaneously measure multiple indices related to the specimen (for example, indices related to physiological activity when the specimen is a plant) using the laser light.

[0028] Furthermore, by providing a step of reflecting the laser light in the second direction as reflected light, the reflected light can be transmitted to the vicinity of the specimen.

[0029] Furthermore, by providing a step of focusing the reflected light toward the vicinity of the specimen, it is possible to focus almost all of the reflected light reflected in the second direction near the measurement site of the specimen, thereby enabling measurement of each site of the specimen with high accuracy and sensitivity.

[0030] Furthermore, by including a process for detecting the degree of polarization, which is the amount of deviation from the optical axis of the reflected light that has passed through the specimen storage section, it is possible to obtain information on dynamic processes, such as material transport and biochemical reactions, on the specimen surface from changes in the refractive index gradient caused by the temperature gradient and concentration gradient near the surface of the specimen through which the laser light has passed.

[0031] Furthermore, when the reflected light is transmitted near the specimen, the first fluorescence and the second fluorescence are emitted by excitation of fluorescent molecules such as chlorophyll contained in the specimen and fluorescent probe molecules in the culture medium. By providing a step of transmitting the first fluorescence in a third direction and reflecting the second fluorescence in a fourth direction, when the specimen and the medium near it contain two different types of fluorescent molecules, these fluorescence can be transmitted and reflected in different directions, respectively.

[0032] Furthermore, by providing a step of detecting the fluorescence intensity of the first fluorescence transmitted in the third direction, it is possible to obtain information on the fluorescence intensity of the first fluorescence.

[0033] Furthermore, by providing a step of detecting the fluorescence intensity of the second fluorescence transmitted in the fourth direction, it is possible to obtain information on the fluorescence intensity of the second fluorescence. [Effects of the Invention]

[0034] The specimen measurement device and specimen measurement method according to the present invention are capable of simultaneously measuring multiple indicators relating to the physiological activity of living organs, including plants, and thus understanding the state of the living organism in real time. [Brief explanation of the drawings]

[0035] [Figure 1] 1 is an overall schematic diagram of a specimen measurement device according to an embodiment of the present invention. [Figure 2] FIG. 2 is an enlarged view of a main part of a holder portion according to an embodiment of the present invention. [Figure 3] 1 is time-series data showing the measurement results of Example 1. [Figure 4]10 is time-series data showing the measurement results of Example 2. [Figure 5] 10 is time-series data showing the measurement results of Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0036] Hereinafter, embodiments of the present invention will be described with reference to the drawings to facilitate understanding of the present invention.

[0037] First, a specimen measurement device 1 according to an embodiment of the present invention will be described with reference to Figure 1. The specimen measurement device 1 is mainly composed of a specimen storage section 10 that stores a specimen S (a plant in this embodiment of the present invention) to be tested, a holder section 20 that holds the stored specimen S in a predetermined position, a laser oscillator section 30 that emits laser light of a predetermined wavelength, a first mirror section 40 that reflects the laser light emitted from the laser oscillator section 30, a condenser lens section 50 that collects the reflected light, a polarization detector section 60 that detects the amount of deviation from the optical axis of the reflected light that has passed through the specimen storage section 10, a second mirror section 70 that transmits or reflects fluorescence emitted from the specimen S or the culture solution in the specimen storage section 10, and a fluorescence detector section 80 that detects the fluorescence intensity of the fluorescence that has passed through or reflected from the second mirror section 70.

[0038] The specimen holding section 10 is substantially rectangular parallelepiped-shaped, with at least two opposing surfaces being transparent so that the laser light emitted from the laser oscillator 30 can pass through from one side to the other. A holder section 20 is installed within the specimen holding section 10 to fix the specimen S to be measured at a predetermined position. A culture solution (e.g., a culture solution containing a Ru(II) complex) is stored within the specimen holding section 10 up to a predetermined water level, and the specimen S is fixed by the holder section 20 while immersed in the culture solution.

[0039] Here, the specimen storage section 10 does not necessarily have to be rectangular parallelepiped, but may have any shape as long as the specimen S can be stored therein and the laser light can be transmitted from one side to the other.

[0040] 2 is an enlarged view showing one embodiment of the holder unit 20. As shown in Fig. 2(a), the holder unit 20 is generally L-shaped in front view and is composed of a vertical unit 21 and a horizontal unit 22. The vertical unit 21 (or other parts of the holder 20) is fixed to a three-dimensional fine adjustment device such as an XYZ microstage (not shown), and the specimen S is placed at a predetermined position on the horizontal unit 22. The horizontal unit 22 is a thin transparent plate (e.g., a glass plate or an acrylic plate), and has a through-hole 23 formed in the approximate center in plan view, penetrating from one side to the other side.

[0041] The shape of the holder portion 20 does not necessarily have to be approximately L-shaped, and can be any shape as long as the holder portion 20 can be fixed to a three-dimensional fine adjustment device such as an XYZ microstage at a predetermined position within the specimen storage portion 10.

[0042] When placing the specimen S in the holder section 20, the portion of the specimen S that is to be measured, for example, if the specimen S is a plant and the leaf of the plant is to be measured, the leaf is positioned above the through-hole 23 on one side of the horizontal section 22. Then, a portion of the leaf enters the through-hole 23, and the specimen S is fixed by being pressed vertically downward with the pressing section 24 made of a glass ball until the portion of the leaf that entered the through-hole 23 faces the other side of the horizontal section 22 in a convex shape.

[0043] By fixing the specimen S to the holder part 20 as described above, a part of the leaf to be measured becomes convex from the horizontal part 22 pointing vertically downward, and the position of the holder part 20 is adjusted using a three-dimensional fine adjuster such as the XYZ microstage described above so that the laser light (reflected light) passes near the bottom of this convex part (a position close enough to not hit the leaf).

[0044] 2(b), the horizontal portion 22 may be curved vertically downward at a predetermined curvature. By curving the horizontal portion 22 vertically downward in this manner, the convex shape of the leaf portion to be measured on the specimen S is further emphasized, thereby further improving the accuracy of transmission of the laser light to the vicinity of the portion of the specimen S to be measured.

[0045] Here, the pressing portion 24 does not necessarily have to be a glass ball, but can be made of any material as long as it is transparent so as not to interfere with the transmission of light for plant photosynthesis from the light source L and is capable of pressing the leaf portion of the specimen S with a certain weight.

[0046] 2(c), a cylindrical body 25 having a predetermined inner diameter may be placed on the horizontal part 22 as the holder part 20. The cylindrical body 25 is made of the same transparent glass or acrylic material as the horizontal part 22 so that the laser light can pass through, and the total length of the cylindrical body 25 is configured to be shorter than the total length of the specimen S by a predetermined amount.

[0047] Then, the specimen S is inserted into the cylindrical body 25. At this time, since the total length of the specimen S is longer than the total length of the cylindrical body 25 by a predetermined amount, a part of the specimen S forms a curved surface and is exposed from both ends of the cylindrical body 25. Then, a three-dimensional fine adjustment device such as an XYZ microstage can be used to adjust the vicinity of this curved surface so that laser light can be transmitted.

[0048] Although the holder portion 20 shown in FIG. 2(c) is cylindrical, it may be any shape as long as it is a cylindrical body having a cavity inside which the specimen S can be accommodated.

[0049] The laser oscillator 30 is a device that emits continuous wave (CW) laser light or laser light having a predetermined pulse waveform. The laser oscillator 30 includes a control device (not shown) and a CW or pulse generator, and the control device sets a target CW or pulse waveform, which in turn generates a CW or pulse waveform having a predetermined output. In an embodiment of the present invention, the laser light emitted from the laser oscillator 30 is used as probe light for polarization measurement of the specimen S and excitation light for fluorescence measurement, as described below.

[0050] When the laser oscillator 30 receives a predetermined timing signal transmitted from a CW or pulse generator, it emits a laser beam having the desired CW or pulse waveform. The laser oscillator 30 is equipped with an optical resonator filled with, for example, argon gas, and is configured to amplify the light in this optical resonator by stimulated emission in the argon gas to emit laser beam.

[0051] In an embodiment of the present invention, it is preferable to use laser light emitted from the laser oscillator 30 in a wavelength band (e.g., 400 to 450 nm) that can excite the fluorescence of chlorophyll in the plant that is the specimen S. However, the laser light is not necessarily limited to this wavelength band, and can be changed as appropriate depending on the type of specimen to be measured, the measurement site of the specimen to be measured, the indicators to be obtained by measurement, etc.

[0052] The first mirror unit 40 is a dichroic mirror that reflects only laser light in a specific wavelength band and transmits light of other wavelengths, and has the function of reflecting the laser light emitted from the laser oscillation unit 30 in a first direction toward a second direction where the specimen S is placed. The measurer can adjust the position of the first mirror unit 40 as appropriate so that the reflected light is reflected in an appropriate direction.

[0053] The condenser lens unit 50 is installed on the optical axis along which the reflected light reflected by the first mirror unit 40 is transmitted, and is a device for guiding the laser light to the specimen S installed in the specimen storage unit 10. The condenser lens unit 50 uses, for example, an objective lens, and the beam of reflected light incident on the objective lens is condensed by the condenser lens unit 50 and emitted toward the vicinity of the measurement site of the specimen S (for example, the roots, stems, or leaves of a plant).

[0054] Next, we will explain the measurement device that measures each index related to the physiological activity of a specimen. First, we will explain the polarization detection unit 60. In the physiological activity of a plant, oxygen, carbon dioxide, nutrients, waste products, etc. move in and out of the plant surface. This movement creates a concentration gradient of physiologically active substances near the plant surface, which changes over time. In addition, various chemical and biochemical reactions occur in plants, and due to the heat of reaction, a temperature gradient also exists near the plant surface, which changes over time.

[0055] These concentration and temperature gradients induce a refractive index gradient near the plant surface, which in turn causes the laser light to be deflected. Utilizing this principle, the polarization detector 60 can analyze information on the dynamic process of the inflow and outflow of physiologically active substances near the surface of the specimen S and their chemical and biochemical reactions from the time-varying deflection signal of the monitored laser light.

[0056] The deflection detection unit 60 is composed of a lens system 61 and a position detection unit 62. The reflected light that has passed through the specimen storage unit 10 is focused by the lens system 61 onto the position detection unit 62, and the deflection at that time is detected by the position detection unit 62. Note that a one-dimensional position sensor or a two-dimensional position sensor is used as the position detection unit 62, but from the viewpoint of improving detection accuracy, it is preferable to use a two-dimensional position sensor.

[0057] Here, it is not necessary to use a position sensor as the position detection unit 62, and for example, a two- or four-division photodiode may be used to measure the deflection.

[0058] The fluorescence detection unit 80 has a function of detecting the intensity of fluorescence excited by the laser light. In this embodiment of the present invention, the laser light is adjusted to pass through a position close to the surface of the specimen S to be measured, so that fluorescent molecules such as chlorophyll close to the surface of the plant that is the specimen S are excited by the laser light, and fluorescence originating from the specimen S, including chlorophyll, is emitted.

[0059] Furthermore, when the laser light passes near the surface of the specimen S, the fluorescent probe molecules in the vicinity of the surface of the specimen S are also excited. The fluorescence of the fluorescent probe is then partially quenched by oxygen, and by measuring the degree of fluorescence quenching of this fluorescent probe, it is possible to monitor the inflow and outflow of oxygen at the surface of the specimen S.

[0060] Fluorescence from the specimen S excited by the laser light and fluorescence from the fluorescent probes passes through the first mirror section 40 and reaches the second mirror section 70 installed further ahead. The second mirror section 70 is configured as a half mirror that is capable of transmitting part of the fluorescence while reflecting the remaining part.

[0061] The fluorescence intensity of the fluorescence derived from the specimen S and the fluorescence of the fluorescent probe that is transmitted through or reflected by the second mirror unit 70 can be detected by a fluorescence detection unit 80 installed beyond it. The fluorescence detection unit 80 is composed of a first fluorescence detection unit 81 and a second fluorescence detection unit 82. For example, by making the first fluorescence detection unit 81 a fluorescence detector that detects the intensity of fluorescence derived from the specimen S, such as chlorophyll fluorescence, and the second fluorescence detection unit 82 a fluorescence detector that detects the intensity of fluorescence from the fluorescent probe, the first fluorescence detection unit 81 is installed beyond the point where the light passes through the second mirror unit 70, and the second fluorescence detection unit 82 is installed at the reflection position of the second mirror unit 70, it is possible to simultaneously obtain time-series data regarding the fluorescence derived from the specimen S, such as chlorophyll fluorescence, and the fluorescence intensity of the fluorescent probe.

[0062] Here, the first fluorescence detection unit 81 is composed of a combination of an interference filter that can transmit light with a fluorescence wavelength of around 690 nm of chlorophyll or with a fluorescence wavelength of around 690 nm derived from the specimen S, and a photomultiplier tube or a weak light detection element, and the second fluorescence detection unit 82 is composed of a combination of an interference filter that transmits light with a fluorescence wavelength of the fluorescent probe, and a photomultiplier tube or a weak light detection element, but is not necessarily limited to such a configuration.

[0063] Furthermore, when a half mirror that reflects chlorophyll fluorescence and transmits fluorescent probe fluorescence is used as the second mirror unit 70, a second fluorescence detection unit 82, which is a fluorescent probe detector, may be installed at the end where the second mirror unit 70 transmits light, and a first fluorescence detection unit 81, which is a chlorophyll detector that detects the intensity of chlorophyll fluorescence, may be installed at the end where the second mirror unit 70 reflects light.

[0064] Furthermore, in the embodiment shown in FIG. 1, the second mirror unit 70, the first fluorescence detection unit 81, and the second fluorescence detection unit 82 are each configured independently, but the second mirror unit 70, the first fluorescence detection unit 81, and the second fluorescence detection unit 82 may be housed in a single housing and configured as an integrated fluorescence detection device.

[0065] The dissolved oxygen (DO) concentration in the vicinity of the specimen S is calculated from the fluorescence intensity of the fluorescent probe detected by the second fluorescence detection unit 82 using the following Stern-Volmer equation (Equation 1): In the following equation, F0 is the fluorescence intensity when DO is not present, F is the fluorescence intensity when DO is present, and K sv is the Stern-Volmer coefficient, C DO is the DO concentration in the vicinity of the sample S.

[0066]

number

[0067] A chlorophyll fluorescence measurement unit 90 using pulse-assisted modulation (PAM) is installed near the side wall of the specimen storage unit 10, and parameters such as the quantum yield of photosystem II in the specimen (for example, the maximum quantum yield F in the respiration process) are measured. v / F m , the effective quantum yield F in the photosynthetic process v ' / F m ') is measured.

[0068] Furthermore, a temperature sensor 100 and a DO sensor 110 are installed in the culture solution stored in the specimen storage section 10, respectively, to measure the temperature and DO of the culture solution.

[0069] The specimen measurement device 1 configured as described above is housed in a dark box B when measuring a specimen, and a light source L that can be switched on and off is installed above the specimen storage section 10. Then, by turning on the light source L, an index of physiological activity in the photosynthetic process of the specimen S is monitored, and by turning the light source L off, an index of physiological activity in the respiratory process of the specimen S is monitored, thereby making it possible to simultaneously obtain all indexes related to the physiological activity of the specimen S as time-series data.

[0070] Next, an embodiment of the specimen measurement device 1 according to the present invention will be described.

[0071] [Example 1] In Example 1, each index of an aquatic plant (aquatic plant Anacharis) was measured as the specimen S. As a preliminary preparation, a culture solution (water or 10 -4 MCuSO4 aqueous solution, or a 1000-fold diluted chemical fertilizer aqueous solution) was added.

[0072] Next, the aquatic plant Anacharis was fixed in the holder 20 and immersed in the culture solution to culture the aquatic plant. A laser beam with a wavelength of 450 nm was passed near the leaves of Anacharis, and the polarized signal, the fluorescence of the Ru(II) complex, the chlorophyll fluorescence of the leaves of Anacharis, and the quantum yield of photosystem II (maximum quantum yield F in the dark) were measured. v / F m、 In the photosynthetic process, the effective quantum yield F v ' / F m The DO and temperature changes in the culture solution were monitored simultaneously for approximately 3 hours. The monitoring period was 3 hours, with the light source L for photosynthesis turned off for the first 1.5 hours and turned on for the next 1.5 hours.

[0073] Figure 3 shows the measurement results when the culture solution was water. Figures 3-A to 3-E show the results of real-time monitoring of the polarized light signal, the fluorescence intensity of the Ru(II) complex, the fluorescence intensity of chlorophyll, the quantum yield of photosystem II, and the DO and temperature of the culture solution, respectively. Figure 3-F also shows the dissolved oxygen concentration near the leaves calculated from the fluorescence intensity of the Ru(II) complex. For comparison, Figure 3-F also shows the DO measured with a DO sensor in the culture solution.

[0074] As can be seen in Figure 3-A, when the photosynthetic light source L was OFF and ON, the time changes in the polarization signal were reversed during plant respiration and photosynthesis. This reflects the fact that the inflow and outflow of physiologically active substances near the leaf surface during respiration is the exact opposite of that during photosynthesis. Furthermore, Figure 3-B shows that the Ru(II) fluorescence intensity increased over time during respiration, but decreased during photosynthesis. This is because oxygen was absorbed and consumed by the leaves during respiration, causing the DO concentration near the leaves to decrease over time. However, oxygen was produced by photochemical reactions during photosynthesis, causing the DO concentration near the leaves to increase over time. Therefore, considering quenching by oxygen, the results shown in Figure 3-B were obtained.

[0075] Figure 3-C shows the time change in chlorophyll fluorescence intensity of the leaf. Because the laser light passes near the surface of the plant leaf, when part of the laser light comes into contact with the surface of the leaf, chlorophyll is excited and emits chlorophyll fluorescence. From the time change in chlorophyll fluorescence intensity, it may be possible to analyze the level of the leaf's photosynthetic ability and the effects of physiological dysfunction due to environmental stress, and this device was able to measure the time change in chlorophyll fluorescence in real time.

[0076] As shown in Figure 3-C, during the respiration process, chlorophyll fluorescence reached a nearly constant value approximately 40 minutes after irradiation with the laser probe light. Furthermore, when the photosynthetic light source L was turned on, chlorophyll was further excited, increasing the fluorescence intensity. The difference in fluorescence intensity when the photosynthetic light source L was on and off reflected the amount of excitation by the photosynthetic light source L.

[0077] Figure 3-D shows the time course of the quantum yield of photosystem II obtained by the chlorophyll fluorescence measurement unit 90 using pulsed absorption modulation (PAM). v / F m is about 0.6, and F v ' / F m ' was about 0.42.

[0078] Figure 3-E shows the temperature and DO changes in the culture solution. When the light source L for photosynthesis was turned on, the temperature of the culture solution rose slightly, and the DO also increased slightly. These temperature and DO changes may vary slightly depending on the installation locations of the temperature sensor 100 and DO sensor 110.

[0079] Figure 3-F shows the DO concentration near the leaf surface (μm level) calculated using Equation 1 from the fluorescence intensity in Figure 3-B, as well as the DO concentration in the culture solution measured with a sensor for comparison. During the respiration process, DO near the leaf surface decreased over time, but during the photosynthesis process, DO near the leaf surface increased over time. In other words, we were able to measure in real time that oxygen is absorbed and consumed during plant respiration, while oxygen is released from the plant during photosynthesis.

[0080] In the physiological activity of plants, physiologically active substances such as oxygen and carbon dioxide enter and exit the plant surface. This entry and exit causes the greatest change in oxygen and carbon dioxide concentration near the plant surface. As the plant moves away from the surface, the change in concentration of physiologically active substances is diluted by diffusion. Therefore, the measurement results of this device, which measures physiologically active substances such as DO near the plant surface, are more sensitive than when measurements are taken in a culture solution or chamber, which are far from the plant surface.

[0081] [Example 2] In Example 2, the effects of fertilizer or growth promoter were measured using the sample measurement device 1 according to the present invention. Figures 4A to 4E show the time-dependent changes in the polarization signal, the fluorescence intensity of the Ru(II) complex, the fluorescence intensity of chlorophyll, the quantum yield of photosystem II, and the temperature and DO of the culture solution when a commercially available plant culture fertilizer was added to the culture solution used in Example 1. Figure 4F also shows a comparison of the calculated DO near the leaf surface with the DO in the culture solution.

[0082] As shown in Figures 4A and 4B, in the presence of fertilizer, the polarization signal and the fluorescence of the Ru(II) complex showed opposite changes during respiration and photosynthesis. Furthermore, these opposite changes were greater than those shown in Figures 3A and 3B in Example 1. This indicates that the movement of biologically active substances such as chlorophyll in and out of the leaf surface became more active. Furthermore, as can be seen in Figure 4C, even when the light source L for photosynthesis was turned on, the rate of increase in chlorophyll fluorescence intensity was small. This means that almost all of the light source L for photosynthesis absorbed by the leaves was utilized for photosynthesis. In other words, it can be said that the presence of fertilizer improved photosynthetic efficiency.

[0083] Furthermore, from Figure 4-D, the maximum quantum yield of photosystem II during respiration, F v / F m is 0.7, and the effective quantum yield of the photosynthetic process, F v ' / F m These quantum yield values ​​are greater than those shown in Figure 3-D in Example 1, which means that both the quantum yield of photosystem II and the effective quantum yield are improved in the presence of fertilizer, and the photosynthetic reaction is more advanced, resulting in faster plant growth.

[0084] Furthermore, as shown in Figure 4-E, the temperature in the culture solution increased over time, and the DO concentration decreased slightly during respiration and increased slightly during photosynthesis. Figure 4-F shows the DO concentration near the leaf surface calculated from the data in Figure 4-B, and the DO concentration in the culture solution measured with a sensor for comparison.

[0085] Compared to Example 1 (Figure 3-F), the DO changes near the leaf surface in Example 2 were approximately twice as large for both the respiration and photosynthesis processes. This means that the presence of fertilizer greatly promoted respiration and photosynthesis, which are linked to the growth of aquatic plants, resulting in faster plant growth. On the other hand, the change in DO in the culture solution measured with the DO sensor was small. This result demonstrates the higher sensitivity of the sample measurement device of the present invention, and furthermore, the promoting effect of fertilizer could be confirmed with measurements over just three hours.

[0086] [Example 3] In Example 3, the influence of environmental stress on the physiological activity of plants was measured using the specimen measurement device 1 according to the present invention. -4 M Cu 2+ The measured polarization, Ru(II) complex fluorescence, chlorophyll fluorescence, quantum yield of photosystem II, and changes in temperature and DO of the culture solution are shown in Figures 5A to 5E. A comparison of the calculated DO near the leaf surface and the DO in the culture solution is shown in Figure 5F.

[0087] From Figure 5-A and Figure 5-B, 10 -4 M Cu 2+ It was revealed that in the presence of , the polarization signal and the fluorescence change of the Ru(II) complex did not change in the opposite direction during respiration and photosynthesis as in Examples 1 and 2. In particular, the polarization signal began to drop significantly immediately after the start of measurement, reached the maximum detectable value of the polarization detector after about 1 hour, and then continued to drop gradually. This means that there was a significant change in the flow of physiologically active substances in and out of the vicinity of the leaves of the aquatic plants. Furthermore, Figure 5-B shows that the fluorescence of the Ru(II) complex increased during both the respiration and photosynthesis processes. This is due to the fact that the fluorescence of the Ru(II) complex increased during both the respiration and photosynthesis processes. -4 M Cu 2+ It is speculated that an abnormality occurred in the photosynthetic reaction in the presence of

[0088] As shown in Figure 5-C, the time course of chlorophyll fluorescence continued to decrease over time, unlike in Example 1 (Figure 3-C) and Example 2 (Figure 4-C). Furthermore, chlorophyll fluorescence continued to decrease even after the light source L for photosynthesis was turned on. This is because the chlorophyll in the leaves was 10 -4 M Cu 2+ It is assumed that some change occurred in the presence of

[0089] From Figure 4-D, it can be seen that both the maximum quantum yield in the respiration process of photosystem II and the effective quantum yield in the photosynthetic process continue to decrease over time, and each value is smaller than that in Example 1 (Figure 3-D) and Example 2 (Figure 4-D). -4 M Cu 2+ This suggests that the photosynthetic efficiency of aquatic plants decreased over time in the presence of α-glucan, inhibiting plant growth.

[0090] As shown in Figure 5-E, the temperature and DO in the culture solution also continued to decrease. Figure 5-F shows a comparison of DO near the leaf surface and DO in the culture solution. -4 M Cu 2+ In the presence of 10 sucrose, DO near the leaf surface decreased over time during photosynthesis. -4 M Cu 2+ It can be said that these had a significant impact on the physiological activity of aquatic plants, causing a significant change in the flow of physiologically active substances on the leaf surface. In other words, it can be said that the aquatic plants were subjected to strong stress from heavy metal ions, which significantly changed their physiological activity.

[0091] Conventionally, stress caused by heavy metal ions had to be determined by long-term growth observation or by measuring the heavy metal content in the plant body, but by using the specimen measuring device according to the present invention, it is possible to determine whether heavy metal ions in the culture solution have caused stress to aquatic plants in just three hours of measurement. Furthermore, in addition to stress caused by heavy metal ions, stress caused by acid rain and eutrophication can also be rapidly measured by using the specimen measuring device according to the present invention.

[0092] In the above description, an embodiment in which the specimen is applied to a plant has been described, but the present invention can also be applied to measurements of other biological organs in which physiologically active substances enter and exit due to physiological activity.

[0093] As described above, the specimen measurement device and specimen measurement method according to the present invention are capable of simultaneously measuring multiple indicators related to the physiological activity of plants, thereby making it possible to grasp the growth state of plants in real time. [Explanation of symbols]

[0094] 1. Sample measurement device 10. Specimen storage section 20 Holder part 21 Vertical section 22 Horizontal section 23 Through hole 24 Pressing part 25 Cylindrical body 30 Laser oscillator 40 First mirror section 50 Condenser lens part 60 Deflection detector 61 Lens system 62 Position detection unit 70 Second mirror section 80 Fluorescence detection unit 81 First fluorescence detection unit 82 Second fluorescence detection unit 90 Chlorophyll Fluorescence Measurement Unit 100 Temperature Sensor 110 DO sensor S specimen L light source B dark box

Claims

1. a specimen storage section capable of storing a specimen to be tested; a holder portion for holding the sample; a laser oscillator that emits laser light in a first direction; a first mirror portion that reflects the laser light emitted from the laser oscillation portion in a second direction as reflected light; a condenser lens unit that is provided on an optical axis of the reflected light and condenses the reflected light toward the vicinity of the specimen; a deflection detection unit that detects a deflection, which is an amount of deviation of the reflected light that has passed through the specimen storage unit from an optical axis; a second mirror unit that transmits the first fluorescence in a third direction and reflects the second fluorescence in a fourth direction, of first fluorescence and second fluorescence emitted by fluorescent molecules derived from the specimen, such as chlorophyll contained in the specimen, and fluorescent probe molecules in a culture solution in contact with the specimen, when the reflected light passes through the vicinity of the specimen; a first fluorescence detection unit that detects the fluorescence intensity of the first fluorescence that has passed through the second mirror unit; a second fluorescence detection unit that detects the fluorescence intensity of the second fluorescence reflected by the second mirror unit. Specimen measurement device.

2. One of the first fluorescence and the second fluorescence is fluorescence from a fluorescent probe, and the other is fluorescence derived from a specimen, such as chlorophyll fluorescence. The specimen measurement device according to claim 1 .

3. A chlorophyll fluorescence measuring unit using pulse modulation for measuring the quantum yield of photosystem II in the specimen is provided near the specimen storage unit. The specimen measurement device according to claim 1 or 2.

4. The specimen is immersed in a culture solution containing a fluorescent probe in the specimen storage section, and an oxygen concentration sensor for detecting the dissolved oxygen concentration of the culture solution and a temperature sensor for detecting the temperature of the culture solution are installed in the specimen storage section. The specimen measurement device according to claim 1 or 2.

5. The deflection degree detection unit is composed of a lens system and a position detection sensor unit, and the reflected light is condensed on the position detection sensor unit via the lens system. The specimen measurement device according to claim 1 or 2.

6. A light source unit that can be switched on and off is provided above the specimen storage unit. The specimen measurement device according to claim 1 or 2.

7. The holder portion is a transparent plate material having a through hole formed from one surface to the other surface; a pressing unit that presses the specimen from one surface side to the other surface side with the specimen placed on one surface side of the transparent plate material so that a part of the specimen enters the through hole and faces the other surface side in a convex shape. The specimen measurement device according to claim 1 or 2.

8. The holder portion is A transparent cylindrical body that is a predetermined length shorter than the total length of the specimen. The specimen measurement device according to claim 1 or 2.

9. A method for measuring a sample contained in a sample container, comprising: emitting a laser beam in a first direction; reflecting the laser light as reflected light in a second direction; focusing the reflected light toward the vicinity of the specimen; detecting a degree of deflection, which is an amount of deviation of the reflected light that has passed through the specimen storage portion from an optical axis; a step of transmitting the first fluorescence in a third direction and reflecting the second fluorescence in a fourth direction, out of first fluorescence and second fluorescence emitted by fluorescent molecules derived from the specimen, such as chlorophyll contained in the specimen, and fluorescent probe molecules in the culture solution, when the reflected light is transmitted through the vicinity of the specimen; detecting a fluorescence intensity of the first fluorescence transmitted in the third direction; and detecting the fluorescence intensity of the second fluorescence transmitted in the fourth direction. Sample measurement method.

10. the specimen is immersed in a culture solution containing a fluorescent probe in the specimen storage section, detecting the dissolved oxygen concentration in the culture solution; detecting the temperature in the culture solution; measuring the quantum yield of photosystem II of the sample. The sample measurement method according to claim 9.

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