The aim of this procedure is to automate leaf shape measurement for high throughput experiments. This is accomplished by first dissecting the leaves and imaging them with a flatbed scanner. The second step of the procedure is to use the leaf J plugin to detect and measure the leaves.
The third step is to prepare the leaves for microscopy and photograph them using a compound microscope. The final step is to trace sellout lines on the acquired images using a touchscreen tablet. Ultimately, the results can show differences in leaf shape, cell size and cell number through this combination of image analysis.
The main advantage of this technique over existing methods is its speed. It's about six times faster than manual measurement. Individuals knew this method may struggle because proper leaf placement is critical for downstream analysis, and because mounting leaves on slides can be technically challenging.
I first had the idea for this method when I realized we had about 15, 000 leaves to analyze, and doing it by hand would take many months and be incredibly boring, demonstrating the plant dissection technique would be causing neuro zue. A research scientist from my laboratory Begin by preparing 8.5 by 11 inch transparency sheets. Each sheet should be split into rectangular frames, large enough to accommodate the leaves from one plant, taper second transparency sheet to the printed sheet to serve as a cover to prevent the leaves from drying out.
Label with the plant, genotype and growth condition. Dissect the leaves of the 26th dayle plants. Arrange the leaves on the transparency in tactic order from youngest to oldest.
Position the leaves vertically within the frame without touching the border or overlapping other leaves. Otherwise, problems will ensue During analysis, It's important to oriental leaves vertically not touching, frame, not overlapping With the leaves arranged. Sandwich them with the second blank transparency sheet and a rack to keep the leaves flat.
Then generate an image using a flatbed scanner with at least 600 dots per inch of resolution. Begin by installing the plugin. Download image J and drag the leaf jja file into the plugins folder of image J.When image J is used, the leaf J plugin will now be available.
Proceed with opening the leaf image file in image J.Make sure image J is version 1.45 s or later. Split the image into three color channels, red, green, and blue by clicking image color split channels. Next, apply a threshold to the image in the blue channel.
Now select all of the leaves from one plant using the rectangular selection tool and then select the leaf J plugin. The plugin will now open a box that allows the various conditions of the experiment to be set, such as an experimental group and genotype. Before proceeding, edit these options, then select the conditions for selected leaves in the region of interest and provide a descriptive name.
Press continue and leaf J will automatically outline each leaf with a unique ROI image. J routines fit in ellipse to the blade and calculate the major and minor axes of that ellipse if necessary. Edit the trace lines from using the ROI manager window.
A touchscreen tablet connected as an external monitor can be useful for this procedure. With the boundaries correctly defined, execute the analysis by pressing. Okay, now leaf J outputs the petal length and various geometries of the leaf blade.
Ultimately, export the measurement results and associated information to a spreadsheet. After scanning the leaves, fix them in F-A-A-F-A. A fixed leaves can be stored at four degrees Celsius for up to six months.
Moving forwards an hour or two before imaging the leaves clear the leaves by changing the FAA fixative to chlorohydrate solution. Once cleared, mount leaves on slides with trichomes facing up using 40 x magnification on a compound microscope image, the mesil layer of the center of each leaf on either side of the main vein, avoiding cells near trichomes or veins. To avoid difficulties in image processing, it's important to remove all air bubbles from slides when mounting leaves and also to get in-focus images in the next step.
After collecting the desired images, open one in image J and trace the leaf cell outlines by image JROI manager tool with aid of the touchscreen tablet and a stylus. This does not utilize the LEAF J plugin. Prior to using LEAF J for experimentation, the accuracy and speed of the plugin was compared to that of an experienced researcher.Overall.
Leaf J finished 5.7 more quickly than the manual calculations and comparing the data from the two methods showed that the results were tightly correlated for all measured parameters of the 3, 532 compared. Data points 172 were outliers. Of these outliers, 29 were caused by the LEAF J plugin much fewer than the 143 caused by human error.
During manual measurement, leaf J has since been updated to further reduce the number of errors. Taking confidence in LEAF J measurements, it was used to assay shade avoidance using a mixed effects model. Treatment and leaf number were fixed effects and replicate was as a random effect.
Many parameters were significantly affected by shade treatment. Alternately, some parameters were not significantly affected by shade. Using a tablet assisted imaged analysis of cell parameters, 67 genotypes of Arabidopsis ANA were examined totaling 8, 629 cells from 877 leaves of 224 plants.
The genotypes were then grouped into six classes. Most genotypes have a normal cell size and number. However, several had larger cell size with normal cell number.
While attempting this procedure is important to use randomization and experimental design when you growing your plants, Remember that working with formaldehyde and chlorhydrate can be extremely hazardous. So precautions such as wearing gloves and working in the fume hood should be taken when doing this procedure. After watching this video, you should have a good understanding of how to do high throughput screening of leaf and cell shape phenotypes using your own experimental conditions Following this procedure.
Additional methods like RNA sequencing can be used to answer questions like which genes effectively shape.
