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MAR was developed to increase the resolution of the image for the sliced plant sample. This revised MAR method showed the detailed distribution of 137Cs accumulation in embryos, which indicated that the plumule and radicle, which grow as a meristem of a root or shoot, were protected from the accumulation of heavy elements. Because of daguerreotype imaging, MAR is now hardly used, and the film emulsion is not available. Here, this method was essentially recreated with a revised processing method for both sample preparation and imaging process.
KeywordsMicroautoradiography Microautoradiography method improvement 109Cd 33P 137Cd Microscopic distribution Rice Grain
7.1 MAR Method Developed
Microautoradiography (MAR) is a conventional imaging method based on the daguerreotype that was used to take photographs several decades ago. This technique was applied to visualize the distribution of radionuclide-labeled compounds within a tissue section. Although the distribution of radionuclides could be visualized in greater detail by MAR than by the IP (imaging plate) method because of the high resolution of MAR, IP imaging has become a widely used method because it has greater quantitative ability and sensitivity than MAR. For these reasons, MAR was performed only to obtain finer images and higher resolution of the radionuclide distribution. However, the application of the classical MAR method to plant tissue sections is associated with several difficulties, such as the preparation of thin sections. To overcome these difficulties, we developed a MAR method for imaging plant tissue section cells [1, 2, 3]. This method was applicable to fresh-frozen plant tissue and has two distinct features: (1) the sample was kept frozen from collected tissue for radioisotope detection, making it possible to fix solutes without solvent exchange, and (2) a 1.2 μm thick polyphenylene sulfide film was inserted between the fresh-frozen plant section and the photosensitive nuclear emulsion to keep the section separated from the emulsion before autoradiography was conducted, which significantly improved the quality of the section until microscopic detection, the quality of the MAR image, and the success rate.
There are two types of photosensitive nuclear emulsion preparations in MAR. In one, the photosensitive nuclear emulsion coats the plant section set on the slide to create a slide–section–emulsion layer. However, in this case, coated plant surfaces frequently exhibit irregularity due to the structure of plant tissue and produce a mixture of silver grains and plant tissue in the same microscope field. In our initial procedure, we attempted to improve this type of MAR in terms of overcoming the irregularity by optimizing the microscope manipulation, e.g. the composition of the microscope images sequentially obtained moving in the z-axial direction. However, the shadow of the plant cells and silver grains were not clearly distinguished, and the quality of the MAR images obtained thus far was insufficient for practical use.
In other preparation, the sliced section was mounted on photosensitive nuclear emulsion pasted on a glass surface, presenting a slide–emulsion–section layer and rarely resulting in irregularity. However, this type of MAR method had other problems, principally related to retention of the section during the development process of the emulsion film. In addition, the adhesive film for retaining the fresh-frozen section was found to hamper the autoradiographic procedure. In fact, we also attempted to develop an autoradiograph after the adhesive film was stripped from the sliced section sample while the sliced section remained on the emulsion surface. After several trials, the success rate of sample retention during the development process was found to be insufficient for routine usage.
The emulsion on the glass slide and the section on the adhesive film were individually processed to develop the autoradiograph and tissue image, respectively. This procedure achieved high-quality image acquisition of the tissue section because the tissue section was rescued before the glass slide was exposed to the reagent for the development process. Other advantages of this method are an extremely high rate of sample retention and a wide choice of tissue staining. On the other hand, the disadvantage of this method is that the positioning accuracy could be reduced due to the separation of the section and the photosensitive nuclear emulsion. The insertion of the 1.2 μm thick film created a concern about the masking effect, particularly in detecting very low-energy β-rays. To address this point, a trial experiment was performed using 3H, which emits β particles with a mean energy of only 5.7 keV, and obtained evidence that the β-ray could produce silver grains through the film (data not shown).
7.2 MAR of 109Cd and 33P in a Rice Plant
As an example of MAR images, the 109Cd distribution of nonelongated stems and shoots above the stem of a rice plant was selected. The distribution images of 109Cd and 33P in the cross section of the crown root are shown.
With regard to the 109Cd signals detected in these tissues using an IP, the signal intensity had a linear relationship with radioactivity. Although the MAR images were related to the 109Cd concentrations, there was no linear relationship between the whiteness of the image and the 109Cd amount, since there were sigmoidal dose–response curves, which are the principle of daguerreotype images. Another problem was that it was difficult to compare the blackening quantitatively across slides, since the precise thickness of the emulsion layer formed on the glass slide could differ among slides. When the stripping film with a uniform thickness was commercially available, the quantitative capability of MAR was ensured; however, the film is no longer in production. Therefore, the precise quantitative capability of MAR is now difficult to ensure.
Figure 7.4 shows the distribution of 33P in the crown roots of rice plants after 15 min of absorption. Interestingly, 33P-phosphate produced many silver grains on the sclerenchyma cells and their neighboring cells in the cortex, adjacent to the epidermis and exodermis cells, indicating that phosphate could easily pass through the epidermis and exodermis cell layers. Phosphate remained in the area of the sclerenchyma cells, which were expected to be connected with each other by Casparian strips, and then easily reached the stele without accumulating in the cortex. The success in visualizing 33P after 15 min of exposure demonstrated both the characteristic transport mechanism of phosphate within the root tissue and the high sensitivity of autoradiography, suggesting the high potential applicability of MAR for ion uptake and transport analysis.
7.3 MAR of 137Cs in a Rice Grain
To compare the distribution of Cs with those of the other elements in the embryo of brown rice, SEM/EDS (scanning electron microscopy/energy dispersive X-ray spectroscopy) was performed to acquire the distributions of K, Mg, N, P, Si, and Ca. As shown in the figure, K, Mg, and P showed higher concentrations at the embryo; however, similar to 137Cs, these elements were also concentrated at the scutellum, not at the plumule or radicle.
Since 137Cs behavior in rice plants has attracted attention, especially after the Fukushima nuclear accident, the uptake behavior of 137Cs was studied in comparison with that of K. The differences in the transport characteristics in plants between potassium (K+) and cesium (Cs+) were investigated using their radionuclides 42K+ and 137Cs+. The result is briefly introduced below. A tracer experiment using nutrient solutions supplied with 42K and 137Cs revealed that the ratio of the root’s K+ uptake rate to its Cs+ uptake rate was 7–11 times higher than the K+ to Cs+ concentration ratio in the solution, and the number varied depending on the K concentration in the solution as well as on the growth condition. After entering through the root tissues, the 42K+ to 137Cs+ ratio in the shoots was 4.3 times higher than the value in the roots. However, the 42K+ to 137Cs+ ratio in each leaf did not differ significantly, indicating that the primary transport of K+ and Cs+ in the shoots is similarly regulated. In contrast, among the radionuclides stored in the roots over 4 h, 30% of the 42K+ was transferred from the roots over the following hour, whereas only 8% of 137Cs+ was moved. In addition, within the xylem, K+ was shown to travel slowly, whereas Cs+ passed quickly through the roots into the shoots. Our study demonstrated the very different transport patterns for the two ions in the root tissue, employing 42K+ and 137Cs+.
- 3.Hirose A (2013) Ph.D. thesis. In: The University of TokyoGoogle Scholar
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