Root Scanning Trays. The use of a root scan tray that fits the entire root imager / scanner area has advantages over the use of, for example, Petri dishes:
Overall, using a root scanning tray that fits the entire root scanner area is a more efficient method of root scanning than using petri dishes. Pro tip: Use plastic tweezers to avoid scratching the acrylic scan tray.
Vienna Scientific offers root scanning trays in two standard sizes & custom made:
Scanner trays are manufactured from 3 mm, clear PMMA (acrylic, transparent). Bottom is fully flat on both sides without casting marks. Trays are water proof. Trays are available in the online store in sets of 2 trays per standard size for a price advantage.
Thoroughly rinse your roots, especially removing adhering mineral soil, organic particles, and rhizosheaths. Store washed roots in water-filled beakers or similar containers in the refrigerator (a few days to 1-2 weeks is usually fine, species depending; longer storage may result in loss of root tips and subsequent decay).
Before taking root images, it is possible to stain the roots. The purpose of staining is to increase image contrast, thereby improving measurement accuracy and making it easier to determine root diameter. Staining may be particularly necessary for very white, fine roots (less than ~0.1-0.2 mm in diameter). However, it should be noted that staining is an additional step in the process that requires more time and careful sample handling. The use of staining can be challenging, especially with small plants where the entire root system is scanned, as it can interfere with subsequent chemical analysis of the tissue. To determine the need for staining, it is recommended that a preliminary test be performed by scanning a sample both before and after applying the staining method. The recommended stain for root staining is Neutral Red, which is prepared at a concentration of approx. 0.16-0.20 g L-1 in water.
As an alternative to staining, consider increasing the scan resolution from the standard 300 to 400 dpi to 600 or 1200 dpi - at the cost of much longer scan times. Higher resolutions are desirable, but often not feasible for many samples without running scanners in parallel. The ideal scanning resolution, however, varies depending on the sample type. For most trees and Phaseolus vulgaris roots, for example, a resolution of 300-400 dpi is sufficient. Conversely, Arabidopsis, grasses and Salix sp. roots should rather be scanned at 600 dpi+. Opting for lower resolutions can significantly speed up the scanning process, particularly when dealing with samples that require large trays. To ensure accuracy in measurements, we recommend scanning a sample at different resolutions to determine if reducing the image resolution compromises precision / recognitiion. For larger and non-flatbed compatible '3D' root systems, camera-based imaging system, such as the Benchtop Root Analyser, might thus be an time-efficient alternative. When conducting root length analyses, grayscale images are often employed (tif or jpg formats), as saving images in grayscale format significantly reduces the file size.
During the scanning process, using a scanner with transparency unit, the roots are placed in acrylic root scanning trays filled with ~ 10-15 mm of water. This allows for better arrangement of roots to minimize overlap and crossing. Be particular careful not to align roots parallel to each other, as this may make them not distinguishable for image analysis programs - resulting in faulty results for (larger) root diameter, underestimated area and root length etc. Make sure that some of the root side branches, tips are not "sticking out" of the water - roots should be spread out in 2D as much as possible. Consider to take heavily branched, and "3D" (i.e. not flat) root branches apart for recognition of individual root tip, individual segments in heavily branched root regions etc. Test the image analysing procedure after a few scans to make sure all root segments are recognized individually by software as required. Stay away from the sides of the root scanning tray to facilitate selection of the regions (of interest) to be analyzed. Plastic forceps and extremely fine plastic pipettes may be used as tools for this delicate procedure. (Pointed) metal forceps are not recommended as they easily scratch the acrylic root scanning trays.
For batch image analysis, consider placing the root scanning trays and the roots in them always at the same position on the scanner. Mark this position on the scanner glass (e.g. with tape) when using trays not covering the whole scanner area.
Adequate lighting (already during placement; e.g., using an additional office lamp) and steady hands are beneficial for optimal results.
After scanning, be sure to collect all the imaged roots for drying (when calculating root morphological parameters). A small sieve may be helpful to quickly collect the smallest root segments. Pour the water from the tray with roots into the sieve.