The overall goal of this video is to illustrate a scanner based method of high resolution image capture of the root tropic response in the model plant opsis. To begin this protocol, seeds from the designated seed stock are planted on Petri dishes containing a transparent medium After the seeds have germinated for the designated time period, Petri dishes are removed from the growth chamber, prepared for scanning, then placed in vertically oriented flatbed scanners. Plates are then turned 90 degrees and scanned for a designated time period.
The images are saved on either the computer or a data storage device and used later for image processing. Prior to image acquisition, prepare aga plates containing the two B scanned plant tissue. First, absorb any collected condensation on the lid and sides of the AGA Plate with a kim wipe.
Next, Apply a generous amount of Triton X 100 to prevent future condensation. Finally, finish this process by wrapping the plate with micropore tape to secure the lid and allow ventilation to begin image acquisition. Set up folders for saving the images to the desired location.
Each folder name consists of a compilation of metadata that relates to the information to be scanned. In this example, unique id age, size, and seed stock were data included on the folder names. This metadata can then be copied and pasted into a folder name.
This Protocol assumes that more than one scanner is being used to collect images and provides instructions to connect multiple scanners to one computer. For this reason, three folders are set up for saving images. The next step is to set the timers for a designated collecting time.
Since there is still quite a bit of preparation to do at this point, be sure to set the timers for a little extra time. Next, turn on the first scanner and open the view scan program. Under the input tab, set the source to the correct scanner and the auto repeat dropdown box to none under the crop tab.
Set the preview area to Maximum and press preview. Choose a crop Box that will capture the region of interest and set the preview area to crop box. Then preview the image again.
To save the collected data in the desired location, press the output tab and find the previously made folder names that correspond With each scanner. Turn on the next scanner And repeat the view scan. Set up directions for the other two scanners, making sure to choose the correct folders in which to save the data.
It is also important to go through each tab and make sure all scanner settings are as desired. Our lab has found the following settings work best for the type of image collection being done. However, different types of image collection may require other Settings.
After all settings are Checked, choose the input tab and set the auto repeat dropdown box to continuous on all three Scanners. Place the prepared Plates in the scanners with the seedlings oriented horizontally and temporarily place the felt side of the background against the plates to ensure stability in the scanner. If performing this protocol with a partner have one person turn the plates 90 degrees and immediately the felt sighted background, the other person then presses the scan button and both secure the background to the scanner with a bungee cord.
Repeats this process for scanners two and three. Each image should take about two to three minutes to completely scan Final images will appear as shown. It's a good idea to check the folders in which the images are saving to make sure all are saving properly.
It is ideal to keep the scanners in an area that will be free of disturbances for the designated time scan. It is also prudent to consider the environmental conditions in the scanning area to ensure ideal phenotypic responses. The following images are examples of those collected using the method described in this video.
Panels A and B and C and D are the first and final images from a single scan period. A and B show the full scanned area. While C and D are a cropped region of the scanned area showing a single plate, several inconsistencies can be observed.
Figure A shows variation in germination and in growth trajectory. Image B shows that plates can accumulate condensation while images C and D are considered to be good results due to the robust growth of seedlings and image quality throughout the run. The procedure outlined in this paper fits into its own niche in the world of root imaging in that it is high throughput and high resolution while still being relatively affordable.
An additional benefit of this approach is that it can be easily customized to accommodate the imaging needs of a particular research group. The method is also being customized for imaging of other.
