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Our first target was water, namely, how to obtain a water-specific image nondestructively. Using a neutron beam, we could visualize water-specific images of plants, including roots and flowers, which were never shown before. Each image suggested the plant-specific activity related to water.
We briefly present how to acquire the image and what kind of water image is taken by neutron beam irradiation. We present a variety of plant samples, such as flowers, seeds, and wood disks. It was noted that neutrons could visualize the roots imbedded in soil without uprooting. When a spatial image of the root imbedded in soil was created from many projection images, the water profile around the root was analyzed. Then, fundamental questions were raised, such as whether plants are absorbing water solution or water vapor from the soil, because there was always a space adjacent to the root surface and hardly any water solution was visualized there. The roots are in constant motion during growth, known as circumnutation, and it is natural that the root tip is always pushing the soil aside to produce space for the root to grow. If the roots are absorbing water vapor, then the next question is about metals. Are the roots absorbing metal vapor? Since we tended to employ water culture to study the physiological activity of plants, the physiological study of the plants growing in soil was somewhat neglected. Later, when we could develop a system to visualize the movement of element absorption in a plant, there was a clear difference in element absorption between water culture and soil culture.
KeywordsNondestructive water image Neutron beam image Water-specific image Root image in soil 3D water image 3D root image Water in flower Water in wood disk Water absorption in seeds
1.1 Neutron Beam Imaging
When collimated neutrons are used to irradiate the sample, the neutrons penetrating the sample can produce images on film or transmit them to a computer through a CCD camera based on the intensity of the neutrons. The neutron beam image can be expressed as a kind of shade if it can be compared to shade images of light. To obtain a high-resolution neutron image, a parallel beam with high quality is needed, which is primarily decided by the features of the neutron collimator. When the length of the collimator is L and the aperture size of the collimator is D, the resolution of the image is reciprocal to L/D. To increase the resolution, it is preferable to use collimators with an L/D of more than 100. Another way to increase the resolution is to reduce the distance between the sample and the film or a camera. The resolution of the imaging time is dependent on the neutron flux and reciprocal to (L/D)2. Another factor that can deteriorate the resolution is movement of the sample itself during the imaging. In the case of plant samples, the development of the tissue is not so fast; therefore, the imaging time is not as critical compared to the case of samples of engineering substances, such as imaging the formation of bubbles in the hot water in a metal pipe.
As shown in Fig. 1.1, the attenuation coefficient of some light elements, including hydrogen, as well as rare earth elements, is extremely high, 100–1000 times higher than those of the other common elements. Because of the selective nuclear reaction of the neutron beam, when the neutron beam is used to irradiate the samples, including light elements or rare earth elements, the beam intensity is drastically reduced after penetrating the sample, which resulted in a clear image of these elements in the sample. In the case of X-rays, the beam reacts with electrons occupying the outside of the nuclide of the element. Since the number and density of the electrons increase with the atomic number of the element, the reaction of the X-ray with electrons increases, which results in a higher attenuation coefficient. Therefore, in the case of X-rays, this attenuation coefficient changes by factors, not by order of magnitudes, from lighter elements to heavier elements, which makes it difficult to distinguish the image of a specific element from that of the neighboring other elements.
Then, what can we see inside a plant irradiated with a neutron beam? In addition to hydrogen, there are only minimal amounts of lithium, boron or rare earths to attenuate neutron beams in plants. Therefore, the neutron beam can be regarded as producing a hydrogen image selectively. Since more than 80% of a living cell consists of water, the hydrogen image from plants is almost entirely water. After complete dehydration, there was hardly any neutron image in most of the plant samples. Therefore, we could regard the neutron image as a water-specific image. The acquired neutron image not only showed water distribution but also indicated the morphological development of the tissue itself. The following section shows how neutron beam images of intact plant tissues can give exquisite microscopic images never seen before.
1.2 Water-Specific Images by Neutron Beam
As written above, when a plant is irradiated with thermal neutrons, the neutron attenuation coefficient changes with the water content in the plant, producing different exposure images on an X-ray film. The different exposure images exhibit different levels of whiteness in an X-ray film; therefore, the whiteness of the film indicates changes in the water content in the tissue. This means that the water content can be estimated from the extent of whiteness in the image.
The cassette was set vertically and was irradiated by thermal neutrons from an atomic reactor, JRR-3 M, installed at the Japan Atomic Energy Agency (JAEA) (Fig. 1.2). The total neutron flux was 1.9 × 108 n/cm2.
After irradiation, the aluminum container became radioactive due to 28Al formation, with a half-life of 2.3 m. However, after 15 m, the radioactivity of the sample was reduced to the background level. The plant samples themselves did not become radioactive after irradiation because the total irradiating neutron flux was relatively low. After irradiation, the film was developed, and the image was scanned by a scanner. When these irradiated plants were left to continue their growth, there was no observable effect compared with unirradiated plants.
1.2.1 2-Dimensional Images of Roots
When the growth of roots imbedded in soil could be visualized, we expected to determine new plant activities, since plant physiology has been developed mainly depending on water culture, where the nutritional conditions can be strictly defined. However, plants growing in the field are influenced by soil conditions, matrix features, nutrient conditions, water amount, etc. All of these conditions differ from place to place and depend on season or climate. However, the physiology of plants growing in the field is not well known, and some of the information is from an agricultural perspective, i.e., how to obtain high yield. Is there any relationship between the growth of the aboveground part and that of the root? Is there any preference regarding the physical condition of the soil for growth? There are many questions to be solved for the plants growing in soil from the perspective of plant physiology. The visualization of roots imbedded in soil could be visualized, provide clues to solve these questions. Therefore, we tried to visualize what we usually cannot see by utilizing neutrons. The following is an example of the root images in soil taken by a neutron beam.
The neutron image of the root imbedded in soil provides two pieces of information: the morphological development of the root and the water profile in the soil. As shown in the figure, we could visualize how the roots developed after the 8th day and 15th day of germination. The white dots shown in the main root indicate that a side root grew from this site and was superposed on the main root.
To discern the water content of the root more clearly, a part of the magnified root of the soybean plant 8 days after germination is shown. The three-dimensional image was acquired by processing the 2D figure of the root, and the height of the image corresponds to the whiteness and the amount of water. The 3D image showed more clearly that the water in the vicinity of the root, where the secondary root was developing, was taken up actively by the root, as shown by the clear emergence of the secondary root.
The second example of neutron imaging applied to the roots is the evaluation of water-absorbing polymers. In water-deficient areas, such as semiarid areas, how to keep water in the soil is a crucial problem, and sometimes special pottery containers filled with water are placed near the roots in the soil so that water gradually seeps out from the container and provides water to the plant for a considerable time. Therefore, water-absorbing polymers have attracted attention as replacements for such pottery containers. Since there are several kinds of water-absorbing polymers with different abilities to absorb water, two kinds of polymers were selected, and the method of supplying water from the polymer to the root was evaluated using neutron images.
In the case of the polyacrylic water-absorbing polymer, as shown on the lower side of the images in Fig. 1.8, the size and whiteness of all the polymers were the same throughout the days of imaging, indicating that the water content in the polymer was not changed. That is, the plant absorbed water only from the soil, and the water in the polymer was not supplied to the plant. Then, a shortage of water occurred, and the roots did not develop well. The length of the main root was approximately the same in all the images.
The upper images in Fig. 1.8 are neutron images of a soybean seedling grown in soil containing polyvinyl alcohol copolymer. At first, there is much water in the soil, in contrast to the observations of the polyacrylic polymer, where water in the soil was absorbed quickly from the first stage. Then, the size of the polyvinyl alcohol copolymers, especially around the upper part of the root, decreased with root development. When the color of the soil and the polymer was compared, first, the soil became darker, and then the polymers became darker and decreased in size. This phenomenon indicated that water was supplied first from the soil and then from the polymer. The side root was well developed compared to that in the polyacrylic polymer in soil. After neutron images were taken, the roots were removed from the container, and the state of the roots was compared. As shown in Fig. 1.8, the roots, including side roots, did not grow well, and the color turned dark when polyacrylic polymer was applied, whereas when polyvinyl alcohol copolymer was supplied to the soil, the roots firmly penetrated the polymer, indicating that the roots searched for water and absorbed water from the polymer.
1.2.2 3-Diimentional Images of Roots
To study the water absorption of the root in more detail, a 3-dimensional (3D) neutron beam image was constructed. The neutron beam image was a 2D image as long as the neutron beam came from one direction of the collimator. When many 2D projection images are taken, with neutron irradiation applied to the sample from different angles, 3D images can be constructed from many 2D images through computer processing. Therefore, the plant was fixed on a rotating table, and the angle of irradiation to the sample was varied. Many 2D images were taken, and then a spatial image was constructed through a computer.
22.214.171.124 3D Image Construction
A five-day-old soybean seedling was transplanted to an aluminum container (35 mm φ, 150 mm) packed with Toyoura’s standard sand containing 15% (w/w) water. The soil surface was covered with aluminum foil to prevent water loss due to evaporation. The samples were kept at 26 °C under 70% humidity with 20,000 lux of light in a growth chamber and were taken out periodically for imaging. These procedures were the same as those to acquire 2D images using an X-ray film. The exposure was performed in a JRR-3 M research reactor installed at the Japan Atomic Energy Research Agency (JAEA). The neutron flux used for the exposure was 1.5 × 108 n/cm2 s.
The size of the image transferred to the computer was 1000 × 1018 pixels. Then, each figure was corrected using two background images. One of the background images was taken when neutrons were absent (dark current), and the other was the image without the sample (shading). Therefore, at each image, dark current image subtraction and shading correction were performed. Then, the root image, 600 × 1018 pixels, was cut out from the corrected figure to reconstruct the CT images. After sectional CT images were constructed at several root heights, sagittal CT images that included the main root were also constructed from a set of sectional CT images.
126.96.36.199 Water Movement Around the Root
Since plant development is slow, CT images can be used to evaluate the “static” spatial water distribution inside and around roots. The following is the spatial water distribution constructed from CT images in a soybean plant.
Since there are no data on how much water exists at the surface of the root, especially within 1 mm of the root surface, the neutron images shown in Fig. 1.12 were of considerable interest. The evidence of the images that there is hardly any water adjacent to the root surface raises many questions about the water absorption activity of the root itself. How does this phenomenon happen? It is widely known that the composition ratio of the soil matrix, air, and water in cultivated soil for farming is approximately 1:1:1. Too much water in soil is harmful to plant growth. Roots absorb water from the soil, but why does hardly any water exist immediately adjacent to the root surface?
One of the answers is that growing roots perform movement, called circumnutation (see Chap. 8). Therefore, roots are always searching for favorable conditions in soil to grow, not only for nutrients or water but also for favorable physical conditions of the soil, hard or soft. Because of this movement of the root, the soil around the root tip is put aside during growth, and as a result, a small space is created around the root tip that seems to facilitate growth.
By stacking the dissection images taken every 50 μm, a 3D image of the water and the root imbedded in the soil was constructed. Figure 1.12 shows the CT image of the root imbedded in soil and the 3D image when approximately half of the area of the horizontal CT images was superposed, showing the root.
There was an obvious increase in side root growth around the upper part of the container (up to 20 mm down from the air/soil interface) compared to side roots located farther down the container. This correlates to a decrease in the water amount found in the soil. However, from this interval of imaging, it was difficult to know whether the decrease in water around the root was due to the horizontal movement of water or to vertical movement along the main root, since water deficiency in some region drives the movement of water toward this site.
Root surfaces and volumes were also calculated from the neutron images. The toxicity of Al ions was analyzed from these calculations since, as previously noted, the presence of aluminum ions is one of the main factors inhibiting plant growth in acidic soil. When 10 mM AlCl3 solution was applied to the soil where a soybean seedling was growing, both the root surface area and root volume decreased . The toxicity of the heavy metal was also visualized by the morphological development of the roots; an example is shown above in Fig. 1.7.
Since neutron imaging has a wide range of effective applications, we tried to apply this technique to other fields. One is fertilizer development, especially in the case of capsule fertilizer, where nutrients seep out gradually during growth (data not shown). Another promising application of this imaging is to study the relationship between root development and yield. It is empirically known that plants with high yield also have well-developed roots. To support this phenomenon with scientific evidence, further study as to determine how water or fertilizer application promotes growth is desirable, since how to increase crop yields is an important issue all over the world.
The water profile around the root is not well known since methods of water measurement in small areas of the soil have not been well developed. In field studies, a lysimeter has been used to analyze the moisture balance in the soil, supplying water to the restricted area, and the changes in water amount due to evaporation along with the soil weight were analyzed for calibration. More specifically, water sensors were inserted into soil at certain intervals from the root, and the water amount at the surface of the root was calculated by extrapolating the water profile measured from the sensors, assuming that there was a gradient of water amount toward the surface of the root.
There is no other method comparable to neutron beams to image the water absorption activity of a living root imbedded in soil. Since the roots are the basic tissue that supports plant activity, nondestructive visualization of the morphological development of the roots and of water absorption has a high potential to be applied not only for physiological research on plants but also to the in situ analysis of plants grown in the field.
1.2.3 Water Images of Flowers
When neutron imaging techniques were applied to plant research, first, this method was mainly employed to analyze the aboveground portion of a plant. For example, neutron imaging of a cowpea plant revealed the role of special internode tissue, whose function was to store water (see Chap. 2, Sect. 2.2). Under water-deficient conditions, water is primarily moved from the internode to other tissues. The neutron imaging method was further developed to measure the amount of water actually moving in an internode using 15O-labeled water (see Chap. 2, Sect. 2.3).
As an example of water analysis in a cut flower, a rose image is introduced. An important issue in the cut flower industry, especially for roses, is how to extend the flowering stage. In the case of a rose flower, sometimes the “bent neck” phenomenon occurs during the shipping of the cut flower. Once this phenomenon occurs, the bent neck never returns to the straight position and withers. The bending phenomenon always occurs at the stem, very close to the bottom part of the flower, and was hypothesized to be induced by water deficiency at this part of the stem. To determine from which regions the rose flower loses water, neutron imaging was employed.
To analyze the water decrease in more detail, neutron images of the flower were taken after 1, 2, 3, 4, 8, 12, 16, and 20 h of the drying process. Before the drying treatment began, 0 h, the water content in the cut flower was very high at the bottom part of the flower and the upper part of the stem, close to the flower. The bending phenomenon occurs at this part of the stem, which initially contained a high amount of water compared to the lower part of the stem. The water content at this site of the stem was maintained well during the first several hours of drying treatment. However, after 8 h, the water in this part gradually decreased.
Next, after 4 h of drying treatment, when the water in the upper part of the stem was not decreased dramatically, water was resupplied. By supplying water again from the bottom part of the stem, the decrease in water at the flower ceased, and water was gradually restored in the stem. However, water was hardly restored at the highest part of the stem close to the flower. The result that water was not reabsorbed at this site in the stem suggested that this site has a special function in maintaining the flowering stage.
That is, when drying conditions began, this part was resistant to decreasing water; however, once water had decreased, water was hardly restored to this part of the stem. This feature might indicate a plant strategy for survival under drying conditions. It seems to require much energy to proceed to the seed ripening stage after the senescence of the flower. Therefore, when the surrounding conditions were not favorable for the plant to develop seeds, such as under low-water availability, the plant could stop the ripening process by discarding the flower part. Bending the higher part of the stem allowed the plant to spend less energy on further development for seed production. However, this example of neutron imaging of cut flowers involves some speculation.
1.2.4 Water Images of Wood Disks
The green moisture in a wood disk image was shown for the first time by neutron beam irradiation. Image analysis also showed how moisture in wood disks is decreased during the drying process.
There are several features of moisture distribution in the trunk of Sugi (Cryptomeria japonica), which is a popular wood utilized in housing materials in Japan. In many cases, there is a so-called white zone at the inner part of sapwood adjacent to the heart wood, recognized by its whiter color. The white zone consists of several annual rings and contains less moisture than surrounding tissues. Green moisture content, especially in heartwood, differs drastically among cultivars or even among individual trees of the same cultivar. It is not known what causes the difference in green moisture content in heartwood, genetics or environmental conditions where it grows. It was reported that there is a reciprocal correlation between the darkness of the color tone and the moisture content in heartwood, where with increasing moisture content, the color becomes darker. Until the tree is cut down, it is not known whether the moisture content of the heartwood is high. Therefore, it is important to distinguish trees with lower or higher moisture content in the heartwood before cutting them down.
When the green moisture content in heartwood was high, drying took longer. Since residual moisture after drying lowers the quality of the lumber, eliminating moisture completely from the heartwood is one of the serious problems in kiln drying. To study the decrease in water during the drying process, four cultivars of Sugi, 24-year-old 25-Gou, 25-year-old Honjiro, 29-year-old 1-Gou, and 30-year-old Sanbusugi were cut down in the University Forest in Chiba, Faculty of Agriculture, The Univ. of Tokyo. Approximately 60 cm of the log at breast height was removed by a chain saw, and each end of the log was sealed tightly with vinyl sheets to prevent moisture loss due to evaporation. The next day, the logs were taken into an atomic reactor, JRR-3 M, installed at Japan Atomic Energy Institute, and cut further to obtain wood disks approximately 1 cm in thickness immediately prior to neutron irradiation. The diameters of the disks were 16.6, 8.4, 8.6, and 13.7 cm for 25-Gou, Honjiro, 1-Gou, and Sanbusugi, respectively.
The disks were fixed on an aluminum cassette by aluminum tape where a gadolinium converter (25 μm in thickness) and an X-ray film (Kodak SR) were sealed in vacuum. The cassette with samples was set perpendicular to the neutron beam and irradiated for 19 s. Ten minutes was needed to cool down the sample.
After irradiation, the wood disks were kept in a phytotron at 60 °C with 90% humidity to reduce the moisture in the disk. During this drying treatment, the wood disk was periodically removed from the phytotron, and neutron irradiation was performed in the same manner as described above. Then, the X-ray film was developed carefully, and the image on the film was transmitted to a computer through a CCD camera (Hamamatsu Co, 2330).
The results can be summarized as follows. In short, when the water content in heartwood was high, the color tone of the heartwood in the neutron image was whiter, and the water amount in the heartwood remained high during drying treatment, whereas when the water content in the heartwood was low, shown by a darker color in the image, the water in heartwood was more easily lost during the drying treatment. The main part of lumber utilized for manufacturing is the heartwood, and water remaining in the wood after manufacturing will induce fractures or cracks during years of usage. However, there is no information about what causes the difference in water amount in heartwood. Indeed, even neighboring trees of the same cultivar showed different water content in the heartwood; the water content was known only after felling the trees.
In the case of the cypress, white color, indicating a water-rich part, was observed, especially at a few outer annual rings. However, from place to place, the water-rich part penetrated a few annual rings. It was interesting that there was no rigid border for water distribution, i.e., water was not confined within one annual ring but seemed to move across the annual rings. If this image indicated water movement across the annual rings, many questions arose. Since there are many ions dissolved in water, the ions could move with water. The movement of ions indicates information movement associated with water movement. The transfer of information across the annual rings suggests that there is another activity within the heartwood and sapwood, information transfer. There is a method to measure the age of the trees by analyzing 14C activity in the carbon contained at a specific annual ring. Since the decrease in 14C activity correlates well with the number of annual rings actually counted, it seems that the carbon structure in the trunk hardly moves, and only water moves in the structure of the carbon network created within the trunk.
We always wondered why most of the tissue in a tree consisted of dead cells. Most living cells exist, particularly in the outer layers of the trunk, and proliferate to enlarge the structure. To maintain the structure of the trees, large amounts of tissues are needed to produce hard trunks. However, when all tissues consist of living cells, they require a large amount of nutrition and energy. The most efficient way to support the structure might be that most of the trees consist of dead cells that do not require energy. However, information must move within the tree, such as to decide when to start the formation of heartwood or to enlarge the heartwood volume.
All the cells in the layer surrounding the heartwood in the sapwood should become dead cells to join the heartwood year by year. It is known that a few percent of the cells in sapwood are alive. When the heartwood increases, how these cells in sapwood, next to the heartwood, become dead cells is not well understood. Are the few living cells in sapwood killed at the border with the heartwood? Although we are apt to focus on the activities of living cells to study plant activity, the role of dead cells might be taken into account.
Important aspects of the physiological activity of a tree could rely on knowing the water and element distribution inside the wood. However, another perspective is to study the element distribution within a tree. We investigated the element distribution within a tree grown in a tropical rainforest, where annual rings were not formed because the weather is relatively constant throughout the year. Through activation analysis of the elements, the distribution of the ion concentration within the disk showed each ion-specific gradient or pattern (data not shown), suggesting that the transmission of each ion might have a specific role in the trunk; therefore, it seemed that there was specific information transfer activity within the tree trunk.
1.2.5 Water Images of Seeds
To visualize how seeds absorb water from outside and how water moves or accumulates in the seed to germinate, water absorption images of the seeds were taken. Five seeds were prepared for neutron irradiation during the water absorption process: broadbean (Vicia faba L.), corn (Zea mays L. cv. Kou 504), morning glory (Ipomoea nil L. cv. Murasaki), wheat (Triticum aestivum L. cv. Minorimugi), and rice (Oryza sativa L. cv. Norin 61). The seeds were dipped into water, and after 1, 2, and 3 h, they were removed from the water and wiped well to remove the water remaining at the surface of the seeds. Then, the seeds were fixed on an aluminum cassette where an n/γ converter and an X-ray film were sealed in vacuum. The neutron irradiation of the seeds was performed repeatedly in the same way used for the other samples cited above. To visualize the water distribution within the seeds more clearly, the 2D seed image was converted to a 3D image, where the height indicates whiteness, that is, the amount of water.
1.3 Summary and Further Discussion
The neutron images provide water-specific images of plants that are not obtainable with other means. The CCD camera employed had the highest resolution (approximately 16 μm) of any other CCD camera available. One pixel in the figure corresponded to approximately 16 μm; therefore, we considered the resolution of the image to be approximately 16 μm. We expect that a CCD camera with higher resolution will be developed and that the water movement inside single cells might be able to be analyzed by neutron imaging.
The neutron imaging of roots was introduced. Through neutron imaging, morphological development of the roots imbedded in soil and water movement close to the root could be visualized, and not only 2D images but also the construction of 3D images enabled analysis of the morphological pattern of the roots and the water profile in soil.
Image analysis of the roots imbedded in soil showed that the water absorption activity in the roots gradually shifted downward from the upper to the lower part. Additionally, from the images adjacent to the root, the water absorption activity at the specific site of the root increased before development of the side roots. The water amount near the root had a gradient that increased drastically from 1 mm from the root surface toward the root, but the root surface was still not saturated with water. The space around the root surface was clearly observed when a 3D image of the root was created. In particular, the dissection image of the root imbedded in soil showed a water-deficient area in the vicinity of the main root surface, from the upper to the lower part. This suggests that there is hardly any contact between water solution and the root surface, which might indicate that the roots are absorbing water vapor rather than water solution. The root tip is always searching for a favorable location and creating space for the root tip to grow in the soil by circumnutation. In the case of a rice plant, one cycle of root tip rotation was found to take 50 min (see Chap. 8). Therefore, there is always a space in the vicinity of the root surface. If the roots absorb water vapor, the absorption of nutritional elements, including metals, from the roots should be drastically different from that of roots growing in water culture. Are the roots absorbing metal vapor, too?
The orientation of root development is another interesting topic, but it was not discussed much here. How does the root decide the direction of the growth? When VA mycorrhizal fungus was placed in a thin box and neutron images were taken, one of the side roots of a soybean plant developed linearly toward the fungus, which was more than 5 cm from the root (data not shown). Whether the hyphae induced the root pattern or not was not discernible.
The analysis of the morphological development of the root enabled us to evaluate the condition of the soil, especially in the development of soil fertilizer, soil conditioners or supplying devices of water. However, there was no reproducibility in the formation of the root pattern, since it is impossible to prepare exactly the same soil conditions for root growth. Therefore, as one solution to evaluate the soil condition based on root development, we measured the root length after formation of the line profile of the root as an indicator to compare growing conditions.
In the case of flower imaging, the water profile within the stem, bulb or pod could be visualized. Water movement derived from neutron imaging suggests tissue-specific functions for water, such as in stems. The bending neck phenomenon suggested another function of the stem. In the case of a rose plant, all of the stem close to the flower consisted of living cells, whereas in other plants, pith was formed where dead cells were packed. During a water-deficient period, living cell activity might cease and thereby prevent water movement toward flower parts. When the pith consisted of dead cells, it could be assumed that water might move easily by capillary phenomena. It was interesting to note that the function of dead cells might also be taken into account.
Many approaches are attempted to extend flowering; one of them is to change the viscosity of water, such as by dissolving an inert gas, such as Xe gas. When water containing dissolved Xe gas was supplied to a carnation flower, the flower took longer to become senescent, suggesting that the water loss due to transpiration was decreased. How water movement is controlled was proposed to provide clues regarding plant activities.
The visualization of green moisture in a wood disk was the first example. There were some other applications of neutron imaging in wood samples, such as detection of wood discoloration in a canker fungus-inoculated Sugi  or to measure the decay resistance of chemically modified wood , etc. The visualization of drying process of Sugi was presented in this chapter. Because Sugi is a popular type of lumber in Japan for building houses or furniture, and only one species exists. Even among the same cultivar of Sugi, the water content in the heartwood drastically varies. It is not known what causes the differences in moisture content in the heartwood. Water distribution within the wood disk showed a water gradient pattern within each annual ring so that the number of the water peaks was the same as the number of annual rings. In the case of Sugi, there was a difference in water content between heartwood and sapwood, and there were always a few water-deficient annual rings at the boundary between heartwood and sapwood, called white rings. However, the size of the white ring increases as the heartwood and sapwood grow. Then, how is water moved across this white ring toward the heartwood while maintaining the white ring? With the development of the tree, the size of the heartwood increases, and white rings are still formed between the heart and sapwood. That is, the white ring grows and maintains a low amount of water, whereas the amount of water in the heartwood or sapwood is maintained during growth. Is there any system to increase the size of this white ring containing a low amount of water?
There are many possibilities for the application of neutron imaging techniques to study plant samples. The neutron images acquired revealed new aspects of plant activity but raised many questions at the same time. Some of the questions raised were about root activities, but another interesting question was about the function or role of dead cells. The dead cells support the plant activities of living cells, which could be estimated through water images; however, how the dead cells are used efficiently is not known. Another question is how plants control water movement. The movement of water is a kind of engine that induces not only water absorption but also growth. It seemed that water movement was not derived simply from diffusion or osmotic pressure.
Though neutron imaging showed static water images of the plant samples, when successive static images were taken, it was possible to estimate the plant activity based on water movement, since the movement of the plants is rather slow.
Then, in the next section, the real movement of water in a living plant is presented utilizing radioisotopes.
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