Microautoradiography (MAR)

Abstract

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.

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 new method we developed achieved clear separation of the photosensitive nuclear emulsion and the section after exposure by sandwiching a 1.2 μm thick film between them (Fig. 7.1).

Fig. 7.1

Schematic illustration of the revised microautoradiography (MAR) method for fresh-frozen plant sections characterized by the separation process [1]. A 1.2-μm-thick polyphenylene sulfide (PPS) film was inserted between the frozen section sample and the photosensitive nuclear emulsion on the glass slide to easily separate the section and emulsion after several weeks of exposure. Progression of the development and tissue processing separately improved the likelihood of success of MAR, and fresh-frozen plant sections of 5 μm in thickness attached to the adhesive film were prepared

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).

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.

Figure 7.2 shows the distribution of ¹⁰⁹Cd in the rice shoot after 24 h of ¹⁰⁹Cd absorption. When the light image and MAR image were superimposed, the xylem cells (Xy) were found to contain less ¹⁰⁹Cd. ¹⁰⁹Cd was more concentrated in the nodal vascular anastomoses (NVA) and the regular vascular bundles (Rv) than in the enlarged vascular bundles (Ev). The ¹⁰⁹Cd concentration in phloem cells was found to be particularly high, whereas the xylem cells contained only small amounts of ¹⁰⁹Cd. In the leaf sheath, the large vascular bundles produced more silver grains than the small vascular bundles. The ¹⁰⁹Cd MAR image could first provide evidence of the intensive accumulation of ¹⁰⁹Cd in the phloem in the nonelongated stem as early as 24 h after ¹⁰⁹Cd administration to the root. The xylem to phloem transport system mediating ¹⁰⁹Cd transport to the phloem in the nonelongated stem implied a significant impact on cadmium partitioning in rice plants.

Fig. 7.2

Distribution of ¹⁰⁹Cd in rice, nonelongated stem and shoot above the stem [1]. (a) Transverse section of the nonelongated stem including the node stained with hematoxylin. (b) Autoradiograph of (a) showing the localization of ¹⁰⁹Cd. (c) Superimposed image of (a) and (b) after the RGB color in (a) was converted to grayscale and the black color in (b) was converted to red. (d) IP image of (a) with pseudocolor. (e, f) Magnified images of the node tissue corresponding to the open boxes in (c) and (b). (g) Transverse section of the part of the shoot above the stem stained with hematoxylin. The leaves were numbered according to their growth from L3 (old) to L8 (young). The first and second leaves (L1 and L2) were in senescence and depressed. (H) Autoradiograph of (G) of the ¹⁰⁹Cd accumulation in the young growing sink leaves, e.g. the seventh (L7) and eighth leaves (L8). Xy: xylem cells; NVA: nodal vascular anastomoses; Rv: regular vascular bundles; Ev: enlarged vascular bundles; Ph: phloem. The radioactivity of ¹⁰⁹Cd in the phloem (Ph) in the Rv was significantly higher than that in the Ev

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.

Figures 7.3 and 7.4 show the distribution of ¹⁰⁹Cd and ³³P in the crown root of rice plants after 24 h and 15 min of absorption, with two and four metaxylem cells in the middle, respectively. As shown in Fig. 7.3, the ¹⁰⁹Cd signal intensity was low in the cortex tissue inside the sclerenchymatous (S) cell layer. A high silver grain image was observed at the outer circle overlapping with the epidermis, exodermis, and sclerenchyma cells, whereas the cortex tissue contained a small amount of ¹⁰⁹Cd, which was also observed in the IP image. Within the xylem cells in the stele, which had a high concentration of ¹⁰⁹Cd, the degree of blackening was similar to that in the surrounding cells, which could not be distinguished by the IP image. MAR clearly revealed the distribution of ¹⁰⁹Cd at the tissue level with high resolution.

Fig. 7.3

Distribution of ¹⁰⁹Cd in the rice crown root after 24 h of ¹⁰⁹Cd absorption [1]. (a) Transverse section of the root with two metaxylem II (MX II) cells in the middle. (b) MAR of (a). (c) Autoradiograph superposed to (a). Red color was added. (d) IP image of the section adjacent to (a). Pseudocolor was added. The ¹⁰⁹Cd signal intensity was low in the cortex tissue inside the S cell layer. Arrows denote the root epidermis (EP), exodermis (EX), sclerenchymatous (S) cell layers, and metaxylem I and II (MX I and MX II) cells. The arrows denote the same site as Fig. 7.4. The ¹⁰⁹Cd signal intensity was low in the cortex tissue inside the S cell layer

Fig. 7.4

Distribution of ³³P in the rice crown root after 15 minutes of ³³P-phosphate absorption [1]. (a) Transverse section of the root with four metaxylem II (MX II) cells in the middle. (b) Autoradiograph superposed to (a). Red color was added. (c) IP image of (a). Pseudocolor was added. The arrows denote the same site as Fig. 7.3. ³³P-phosphate concentrated in the sclerenchyma cells and their neighboring cells in the cortex, which were adjacent to the epidermis and exodermis cells

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.

3 MAR of ¹³⁷Cs in a Rice Grain

The 3D ¹³⁷Cs image in a rice grain was shown in the previous chapter (Figs. 6.3 and 6.5), where ¹³⁷Cs accumulated in the embryo and the outer bran layer. To acquire a finer image of ¹³⁷Cs distribution in the grain, especially in the embryo, MAR was performed on rice grain containing ¹³⁷Cs. A rice plant was supplied with ¹³⁷Cs solution (200 Bq/mL) for 24 h when flowering was started. Then, after 28 days, the mature brown rice was harvested, and MAR images were acquired. Figure 7.5 shows the distribution of ¹³⁷Cs in the embryo. As shown in the figure, most ¹³⁷Cs accumulated in the scutellum, and a very small amount of ¹³⁷Cs was observed in the plumule, radicle, and embryonic root, suggesting that the tissue with meristems that grew after germination was highly protected from the heavy element Cs.

Fig. 7.5

Distribution of ¹³⁷Cs in the embryo of a rice train partially. Red color was added to the microautoradiography image (MAR) according to the intensity of the image. In the light image under the microscope, P: plumule, S: scutellum, R: radicle (embryonic root). The distributions of K, Mg, N, P, Si, and Ca are shown in grayscale. K, Mg, and P concentrations were high in the embryo; however, they were not accumulated in the plumule or radicle, which is similar to those of ¹³⁷Cs

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⁺.