Studying Cell Death Initiation Using a Digital Microscope

NOTE: Sections 1 and 2 describe a modified protocol for plant material preparation based on the methods outlined by Lukan et al.⁷. Specifically, some modifications to controlled environmental conditions and inoculum preparation were made.

1. Growing potato plants

1. Grow healthy potato cv. Rywal plants in stem node tissue culture⁸.
2. After 6-8 weeks, cut ten 1 cm-long explants containing nodes from potato plants on sterile paper under sterile conditions using sterile tweezers and a scalpel knife.
3. Transfer them to plastic boxes with Murashige and Skoog (MS30) medium (Figure 1A) and grow them under controlled environmental conditions (22 °C in the light and 19 °C in the dark with 250 µE or µmol/m²/s radiation and a 16-h photoperiod, at a relative humidity of 55% ± 5%).
4. Transfer them to the soil 2 weeks after micropropagation.
   1. Fill the pots (r = 10 cm) with soil. Use a finger to create a 3-4 cm deep hole in the soil in the middle of a pot, fill the hole with water, and wait for it to absorb.
   2. Place one plant per pot into the hole, leaving the leaves above the surface. Gently cover the roots with soil (Figure 1B).
      NOTE: Some plants might not develop roots. Exclude those plants from the analysis.
5. Grow the plants in soil in a growth chamber under controlled environmental conditions (22 °C in the light and 19 °C in the dark, at a relative humidity of 55% ± 5%, with 250 µmol/m²/s radiation and a 16-h photoperiod).
6. After 3-4 weeks, plants are ready. Use these plants to be inoculated.
   NOTE: Plants should have at least 3-4 fully developed leaves with visible leaflets (Figure 1C). They should look healthy, with no visible symptoms (yellow or brown leaves), which could be mistaken for lesions caused by PVY. To achieve comparable results, plants should be inoculated at the same time (e.g., at 9 am) in all experiments to avoid possible effects of circadian rhythm on plant immune response and lesion formation^9,10,11.

2. Inoculum preparation and potato inoculation

1. Inoculate the plant with PVY strain N-Wilga (PVY^N-Wi; accession no. EF558545) inoculum as stated below.
   NOTE: More plants can be inoculated in parallel if more than one digital microscope is available. Plants should have at least three fully developed leaves before inoculation (Figure 1C). PVY^N-Wi is multiplied and maintained in potato cv. Pentland.
   1. Prepare phosphate buffer, supplemented with sodium diethyldithiocarbamate (DIECA), for inoculation: Mix 1.3 mL of 0.2 M NaH₂PO₄, 8.7 mL of 0.2 M Na₂HPO₄, and 0.225 g of DIECA. Make up the volume to 100 mL using ddH₂0. Adjust pH to 7.6 by adding 1 M NaOH or 1 M HCl solution.
   2. Harvest 6-8 weeks old PVY^N-Wi infected potato cv. Pentland plants from tissue culture in extraction bags with filter net (0.5 g per 6 plants) and add phosphate buffer supplemented with DIECA (4x mass of plant material, mass: volume ratio). Use a hand homogenizer for 1-2 min to obtain a homogeneous solution.
   3. Lightly dust the first three bottom fully developed leaves of 3-4 weeks old potato plants (from step 1.6) with carborundum powder.
      NOTE: Adjust the amount of carborundum powder. Too much may cause damage, while too little might result in ineffective infection. Optimally, 0.1 mg/cm of carborundum powder is used, which is approximately 1.5 mg of powder per leaf of average size (approximately 15 cm²).
   4. Gently rub the leaves with the inoculum (~100 µL per leaf). After 10 min, thoroughly wash the leaves with tap water.
      1. Be careful not to harm the leaves. Do not exceed the incubation time. Adjust the amount of the inoculum according to leaf size – 6.5 µL/cm, which is approximately 100 µL per leaf of average size (approximately 15 cm²).
   5. Transfer the plants to a growth chamber and grow them under controlled environmental conditions (28 °C in the light and 28 °C in the dark at a relative humidity of 55% ± 5%, with 250 µmol/m²/s radiation and a 16 h photoperiod) for 3 days.

3. Plant preparation and using the digital microscope for recording lesion development

1. Transfer the inoculated plants from the growth chamber maintained at 28 °C to a growth chamber at 22 °C 3 days after inoculation, with other conditions as described in step 1.5.
2. Select a plant for observation. Immobilize the second inoculated leaf using tape (Figure 1D). Make sure that the desired leaf is fully immobilized before starting with imaging (Figure 1D).
3. Install a software application for image capture on the laptop computer. Connect the digital microscope to the computer. Open the software.
   NOTE: Make sure that the plant, digital microscope, and laptop are positioned on the shelf near the socket to plug in the computer. The decision on the selection of leaves to be monitored might depend on the biological question studied, e.g., the type of process that leads to the formation of limited programmed cell death.
4. Adjust the microscope above the immobilized leaf. Focus using the dial on the digital microscope (shown in Figure 1E).
   1. Ensure that the line of sight is as wide as possible to increase the chances of capturing the formation of the lesion but still magnified enough to spot the lesion's appearance (typically, 25x magnification is used).
5. Set the camera settings. Click on the Settings button in the top right corner of the image (Figure 2A, circled part 1) and adjust camera settings: Brightness (60-70), Contrast (10-15), Hue (0), White Balance, Saturation (45-50), Sharpness (0), Gamma (5) (Figure 2B). The settings used in this study were as follows: Brightness (64), Contrast (14), Hue (0), Saturation (47), Sharpness (0), Gamma (5).
   NOTE: Camera settings should be adjusted to outside light to ensure the best image quality. Settings could be adapted according to the user's conditions in the growth chamber.
6. Set the image capture settings by clicking on the icon for Image Capture (Figure 2C, circled part 2). Click on the button Time-Lapsed Video and set Image Capture every 15 min for 24 h (Figure 2C).
   NOTE: The interval between the images taken and imaging duration are fully flexible and could be adapted according to the needs of the experiment. In the time between image capture, the LED light is switched off. The LED light is automatically switched on during image capture.
7. Start image capture by clicking on the START button (Figure 2C).
   NOTE: After being transferred from 28 °C to 22 °C, plants should start to develop lesions within 24 h. If not, this could be a sign of ineffective inoculation. Adjust the amount of carborundum powder, ensure that the time of inoculum incubation on leaves was correct, and determine virus abundance in the inoculum by qPCR.
8. To save images, select all images and click the Save icon (Figure 2B, circled part 3). Set Export Options. Set DPI at maximum (300) (Figure 2D). After saving, delete all pictures in the program.
9. For image analysis, any program for image viewing/editing is sufficient. The usage of free software for image editing, ImageJ, is explained below.
   1. Import the time sequence of images from a single field of vision (on the top left corner, click File, select Import, and then Image Sequence). Paste the path of the directory of saved pictures and press the OK button to start the conversion.
   2. After the conversion, the software automatically opens an internal video player showing the final video. Export the video file by clicking on File > Save as option and selecting AVI Format. A small window opens. Set the Frame Rate as 0.3 fps and press OK to save the video as an AVI video file.
      NOTE: The time of lesion development can also be determined by manually checking all of the images and finding an image where the lesion appears.