Imaging plate

Two-week-old plants at the postbolting stage were used for detection of ²²Na and ⁴³K absorption. For ⁴³K absorption experiments, plants were incubated in 10 ml of hydroponic medium supplemented with 2 MBq of ⁴³K for 15 hours in the light. Then, the plants were washed twice with hydroponic medium without ⁴³K and separated into roots and shoots. For ²²Na absorption experiments, plants were incubated in 10 ml of hydroponic medium containing 75 mM NaCl and 500 kBq of ²²Na (Chiyoda Technol, Tokyo, Japan) for 2 days in the light. To keep the volume of the hydroponic medium constant at 10 ml, fresh hydroponic medium containing 75 mM NaCl was added every 12 hours. Then, the plants were washed with the hydroponic medium containing 75 mM NaCl twice and separated into roots and shoots. The labeled plant materials were then put onto imaging plates (FUJIFILM, Tokyo, Japan) and exposed at 4°C for 8 hours. The imaging plates were scanned in a Typhoon FLA 9500 laser scanner (GE Healthcare Japan K.K., Tokyo, Japan).

X-ray diffraction patterns were recorded at the BL45XU beamline of SPring-8 [18 (link)]. The specimen-to-detector distance was 2.4 m, and the X-ray wavelength was 0.1 nm. As a detector, a cooled CCD camera (C4880-50, Hamamatsu Photonics, Hamamatsu, Japan; 1000 × 1018 pixels, pixel size, 150 × 150 μm) was used in combination with an image intensifier (VP5445-MOD, Hamamatsu Photonics). Some of the patterns from bumblebee muscle fibers were recorded on Imaging Plates (Fuji Film Co., Tokyo, Japan) with a pixel size of 50 × 50 μm. The diffraction patterns recorded under the same experimental condition were summed to improve the signal-to-noise ratio, and the background scattering was subtracted by the method described previously [15 (link), 19 (link)].

Radiographs of proximal tibial metaphyses containing the zirconia implants were recorded on imaging plates (Fuji Film, Tokyo, Japan) using a medical Roentgen diagnostics system (DR-150-1, Hitachi Inc., Tokyo, Japan). An X-ray was irradiated perpendicular to the rectangular plane of the implant using a universally rotating sample holder. The bone-to-implant contact ratio was evaluated for the radiographs by a physician blinded to the sample identifications using Image J ver. 1.48. The bone-to-implant contact ratio was defined as the percentage of the length of direct contact between the bone and the implant surface in the total length of the implant within the bone on X-ray radiographs. The length of direct contact was the bone formed on the surface of the implant placed inside the bone and was the sum of the length that was continuous with the bone cortex and in direct contact with the implant. Moreover, bird’s-eye views were prepared from the radiographs by plotting the intensity of X-ray absorption at each X‒Y point on the Z-axis.

After 1 h, the plants were washed twice with hydroponic medium containing 2 mM K⁺, and dissected with surgical scissors into shoots, hypocotyls and roots. The plant material was put onto imaging plates (Fuji Film, Japan) and exposed for 3–5 h at 4 °C. The imaging plates were scanned in a Typhoon FLA 9500 laser scanner (GE Healthcare Japan K.K., Japan). The plant material was then transferred into individual tubes and the weight was determined.

In order to quantitate the amount of IVA absorbed in the tissues of Arabidopsis seedlings upon bioassays, ¹⁴C-labeled IVA was used for the bioassays, and the radioactivity from Arabidopsis tissue was quantified. Briefly, 10.1 µL of ¹⁴C-labeled IVA corresponding to a 2 mM concentration (American Radiolabeled Chemicals) was mixed with 9.9 µL of 200 mM non-radiolabeled IVA to make 100 mM of ¹⁴C-labeled IVA (corresponding to 50 µCi/mL) and was serially diluted with MeOH to prepare 10-mM and 1-mM solutions. Fifteen Arabidopsis (Col-0) seedlings (5-day-old) were transferred to test plates, and ¹⁴C-labeled IVA mixed with non-labeled IVA was spotted onto the filter paper in a 35 mm dish and incubated for 3 days under a long-day photoperiod at 23 °C. The seedlings were transferred to filter paper and wrapped with plastic film. Imaging plates (Fujifilm, Tokyo, Japan) were exposed for 24 h and scanned with Typhoon FLA-9500 (GE Healthcare, Chicago, IL, USA) and the radioactivity was analyzed and quantitated using Image Quant (GE Healthcare) software. Radioactivity from five seedlings either at the proximal, mid, or distal position were pooled and quantitated. Arbitral units of the radioactivity were converted to DPM by a standard curve. The experiments were repeated at least three times, independently.

The test plant was dissected and placed on paper sheets after the PETIS imaging was finished and then placed in contact with imaging plates (Fujifilm, Tokyo, Japan) in cassettes. After 2 days of exposure, the imaging plates were scanned using a bio-imaging analyzer (BAS-1500; Fujifilm, Tokyo, Japan) to obtain the auto-radiographic images of ⁷⁴As in plant.