A Novel Imaging Protocol for Investigating Arabidopsis thaliana Siliques and Seeds Using X-rays

Materials and reagents

Biological materials

Arabidopsis thaliana ecotype Columbia (Col_0) plant at midflowering, complete flowering, and seed harvest stage (Boyes et al., 2001).

Laboratory supplies

1. Fine precision medium tipped tweezers/forceps (Fisher Scientific, catalog number: 12-000-157)

2. Petri dish (Fisher Scientific, catalog number: FB0875712)

3. Permanent ink markers

4. Paper towels (Fisher Scientific, catalog number: 19-040-898)

5. Tape (Fisher Scientific, catalog number: 15949)

6. X-ray source: Kevex PXS10-16W MicroFocus system

7. Boss LS-1416 CO₂ laser cutter

Equipment

1. Inspex 20i X-ray machine

   For custom manufacturing of the X-ray imaging cabinet, details are provided in Figure 1. The Inspex 20i X-ray machine was assembled by Kodex Inc. (Nutley, NJ).

   
   

   
   Figure 1. Schematics of the X-ray imaging setup. (A) Cartoon sketch of the Inspex 20i X-ray machine with all the accessories. The custom steel cabinet (J) measures 64.77 cm (depth) × 127 cm (height) × 78.74 cm (width) and is lined with a lead sheet to conform to BRH 21 CFR 1020.40 and FDA 21 CFR 1020.40, in accordance with Georgia State X-ray radiation regulations. The X-ray source (G) is a Kevex PXS10-16W MicroFocus system that produces a downward facing cone-beam so that shadows can be detected on the Kodex LTX-1717 digital detection panel (E) located at the bottom of the cabinet. This amorphous silicon flat panel detector has a cesium iodide scintillator in the outer layer that allows for the conversion of X-rays to light, which is then digitized by the photodiode layer below for transmission to the computer for display on the screen. The microfocus system allows focused images to be enlarged or reduced by varying the specimen height between the source and detector (closer to the source results in a magnified image). Placing the specimen as close as possible to the source (G­(a)) results in a field of view of approximately 9.5 mm². The field of view adjacent to the detector (E) is approximately 45 times greater at 431.8 mm² (the detector is 17 × 17 inches). Components external to the cabinet (red): (A) Power supply for the X-ray source; (B) Power supply and data transfer unit for the detector; (C) Computer to render and save images; (D) Uninterruptable power supply and surge suppressor for the X-ray machine. Components inside the cabinet (black): (E) The Kodex LTX-1717 digital detection panel, E(w) width 44.45 cm, E(d) depth 44.45 cm, E(h) distance between detector and source 104.14 cm; (F) The cone beam path of the X-ray (cone of illumination, 53°); (G) The Kevex PXS10 MicroFocus X-ray source (maximum settings of 80 kV, 0.100 mA); (H) Image collection shutter trigger; (I) Touchscreen monitor and keyboard for image viewing. (Average settings for this study: Silique specimens in this study were placed on a low-density plastic platform suspended 98.4 cm above the detector, and the source was operated at 40 kV and 0.09 Amps). (B) Picture of the X-ray machine with the front door open.

   
   

2. Scale for the measurement of siliques/seeds using the Inspex 20i X-ray machine

   We used an X-ray semi-transparent scale that was cut from cardstock (weight 90 Lb) using a Boss LS-1416 CO₂ laser cutter (70 W). We did this because the printed hashmarks on solid plastic or metal rulers are not visible in X-ray images. We designed the scale using the Adobe Illustrator vector drawing package. Only the millimeter hashmarks and the external contour of the scale were cut; the millimeter number markings were not. The kerf width of the laser cutter (aka. hashmark line width) measured an average of 0.152 mm. This was wider than we had hoped, but the centers of the marks were extremely accurate. The dimensional accuracy of the laser cutter was listed as being ± 0.0127 mm, and this high accuracy was confirmed using multiple micrometer measurements from the hashmark centers. Since the scale bars and their hashmarks could be seen beside the seeds and siliques, they proved a simple, economical, and reproducible method of calibrating X-ray image dimensions.

Software and datasets

1. VetView Console: All-In-One Workstation Software is used for image acquisition, processing, and editing.

2. DaVinci Resolve 11: Calibration software, run as a script, to dark calibrate data read from the X-ray detector while the source is not providing illumination.

3. FIJI ImageJ Build 2.13.1: This image processing program was used to count and measure diameters of the seeds within the silique.

4. GraphPad Prism Version 9.4.1: Statistical analyses and production of graphs.

Procedure