To begin, transfer the virus-infected potato plants to a growth chamber maintained at 22 degrees Celsius. Pick a plant for observation, and use adhesive tape to fully immobilize the second inoculated leaf. Next, connect the digital microscope to the computer and launch the image capture software.
Place the leaf under the microscope. Then, use the dial to focus on the surface of the leaf. To adjust the camera settings, click the Settings button.
Now, set the brightness to 64, the contrast to 14, and the hue to zero. Next, adjust the white balance. Then set the saturation to 47, sharpness to zero, and gamma to five.
Click on the image capture icon and select Time-lapsed Video option. Next, set image capture for every 15 minutes for a total of 24 hours. Press the Start button to begin the image capture.
To save the captured images, select all images and click the Save icon. Now, choose the export options. Then, set dots per inch to maximum.
Once saved, delete all images from the program. To edit the images, launch an editing software like ImageJ. Click on File, then select Import, and choose Image Sequence to import the time sequences of images from a single field of vision.
Paste the path of the directory of saved pictures, and press Okay. Once the conversion is complete, ImageJ will automatically launch an internal video player displaying the finished video. Click on File, then Save As, and select the AVI format to export the video file.
When a small window opens, set the frame rate to 0.3 FPS, then press Okay to save the video as an AVI file. Three to four-week-old inoculated potato plants with at least three to four fully developed leaves were used for digital microscopic analysis. The same area on the inoculated leaf was observed at 15-minute intervals to determine the lesion occurrence and expansion over time.
At 14 hours of observation, no lesion was visible. About 90 minutes later, a visible lesion was recorded. The lesion started to expand two hours later, and continued expansion over the next eight and a half hours.
