A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

NOTE: It is important to prevent sleep movement of living leaf samples during time-lapse imaging. To minimize mechanical stress on leaves, gentle fixation of leaves is necessary. Only adequately prepared leaf samples produce time-lapse images suitable for various image analyses. A protocol using transgenic Arabidopsis plants expressing YFP-NLS fusion under the control of PR1 gene promoter (pPR1-YFP-NLS plants) and Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) is described below as an example.

1. Preparation of plants and pathogens

1. Fill a plastic cell plug tray (Table of Materials) with autoclaved soil.
2. Sow one transgenic Arabidopsis seed per cell (Figure 1A).
3. Transfer the tray to a growth room maintained at 23 °C and grow the plants under continuous white light for 2-3 weeks.
4. Two days prior to pathogen inoculation, streak Pseudomonas syringae pv. tomato DC3000 avrRpt2 strain (Pst_a2) from a glycerol stock onto NYG medium (5 g/L peptone, 3 g/L yeast extract, 20 mL/L glycerol, 15 g/L bacteriological agar, pH 7.0) containing 100 mg/L rifampicin and 50 mg/L kanamycin and incubate at 28 °C for 48 h.
5. Harvest the bacterial cells that appear on the surface of the medium using plastic tips, transfer them to a plastic tube containing 10 mM MgCl₂, and resuspend. Measure the optical density (OD) of the solution at 600 nm (OD₆₀₀). Adjust the final cell concentration of bacterial cells to 10⁸ colony formation units (CFU)/mL, which normally corresponds to OD₆₀₀ = 0.2¹¹.

2. Pathogen inoculation

1. Carefully cut out a cell plug containing a 2-3-week-old plant without damaging the plant. Set the cell in an empty cell plug tray (2 x 2 cells are sufficient) to maintain a good balance (Figure 1B).
2. Select a visibly healthy leaf for inoculation. Generally, the third, fourth, and fifth leaves (#3, #4, and #5, respectively, in Figure 1C) from the bottom of the plant are easy to handle. Use leaves at the same position in a set of experiments for better reproducibility. Water the soil holding the plant before inoculation for long-term time-lapse imaging.
3. Optionally, in the case of analyzing stress-responsive promoters such as pPR1, to ensure that plants are not naturally stressed, examine the leaves under a fluorescence stereomicroscope prior to pathogen inoculation to verify the absence of YFP signal. Exclude leaves showing YFP signal from the experiment.
4. Wear disposable latex gloves before infiltration to avoid direct contact with the pathogen. Using a 1 mL needleless plastic syringe, carefully infiltrate the abaxial side of the leaf with the bacterial suspension (10⁸ CFU/mL)¹¹ (Figure 1D). Inoculation of a small portion on one-half of the leaf enables a good visualization of pPR1 activity; the infiltrated area becomes visible as darker green in color compared with the remaining leaf. Be extremely careful not to cause any mechanical damage to the leaf during infiltration.
   NOTE: Make sure that all the intercellular spaces in the infiltrated area are completely (vertically) fulfilled with the pathogen suspension; otherwise, the PCD domain will be difficult to visualize under the fluorescent stereomicroscope. This can be simply confirmed by completion of dark greening in the infiltrated area.
5. Absorb the excess of bacterial suspension from the area surrounding the infiltrated section of the infiltrated leaf with a soft paper towel.

3. Mounting the inoculated leaf

1. Immediately after inoculation, fix a glass slide on the plastic tray using surgical tape (Table of Materials) such that the infiltrated leaf is located at the center of the glass slide. Ensure that the inoculated leaf blade is completely fitted within the glass slide (Figure 2A).
2. Prepare double-layered plastic tape (in the case of 0.2 mm thick tape, see Table of Materials) and cut it into two pieces (Figure 2B; pieces 1 and 2) to fit the spaces along the petiole of the infiltrated leaf. Arrange the length of these two pieces, indicated with a double-headed arrow in Figure 2B, to fit into the length of the double-headed arrow shown in Figure 2A.
   NOTE: Cutting out a corner from each of the two pieces helps avoid damage to the leaf blade (Figure 2B, arrowheads; also see step 3.4 below). Any kind of plastic tape with similar thickness is suitable for making a bridge over the petiole. The stiffness of plastic tape is important for easy handling during the procedure described below.
3. Using a pair of fine tweezers, stick tape pieces 1 and 2 on either side of the petiole such that the cut corners of each piece align with the base of the leaf blade (Figure 2C). Ensure that the tape pieces do not touch the petiole or leaf blade.
   NOTE: These double-layered plastic tape pieces at the base of the leaf blade act as spacers and prevent physical stress on the petiole during mounting.
4. Prepare an additional piece of double-layered plastic tape (Figure 2B, piece 3) to fit the size of the double-headed arrow in Figure 2B and stick it on top of the tape pieces 1 and 2 to form a bridge over the petiole (Figure 2D,E). Be extremely careful not to catch the petiole and leaf blade directly between the tape pieces at positions indicated with arrows in Figure 2D,E.
5. Gently stick a small piece of surgical tape (Figure 2F, piece 4) on the glass slide above the tip of the leaf blade so that the leaf blade is fixed very softly on the glass slide. Only firmly press down the part of surgical tape directly touching the glass slide (Figure 2F, area outlined with a dashed red line), not the other part overlying the leaf.
6. Gently stick another small piece of surgical tape (Figure 2G, piece 5) at the border of the petiole and plastic tape pieces (1, 2, and 3) so that the petiole is very softly fixed onto both the glass slide and plastic tape pieces. Only firmly attach the part of surgical tape directly touching the glass slide to the glass slide and plastic tape pieces (Figure 2G, area outlined with a dashed red line), not the other part overlying the petiole.
7. Prevent neighboring leaves from moving into the field of view of the microscope using 200 µL pipette tips (Figure 2H). Insert the pipette tips in the soil to gently hold the neighboring leaves away from the infiltrated leaf. Be careful not to insert the tips too deep in the soil to avoid possible root damage.
   NOTE: The prepared plant is now ready for fluorescence stereomicroscope imaging.

4. Microscopic time-lapse observation

1. Turn on the fluorescent stereomicroscope.
   NOTE: Here, an automated stereomicroscope equipped with a highly sensitive 1.4 megapixel monochrome digital camera in 12-bit mode is used (Table of Materials). The microscope should be placed in a dark room fitted with an air-conditioning unit or in a dark cabinet with adequate ventilation to maintain the room temperature at 23 °C during time-lapse imaging.
2. Set the plant in the space above under the objective lens of the stereomicroscope for imaging.
3. Set up the parameters for time-lapse imaging (see examples in Table 1). Make sure to program steps for light exposure during the interval period of time-lapse imaging since light has a major impact on plant immunity¹².
   1. Use the conventional YFP filter (excitation 500-520 nm; emission 540-580 nm) to visualize the YFP signal.
   2. Use the Texas Red (TXR) filter (excitation 540-580 nm; emission 610 nm long pass) to visualize chlorophyll autofluorescence so that the PCD domain is visible as a dark area (no autofluorescence) surrounded by YFP-positive cell layers⁹ (Figure 3A).
   3. Use the conventional epi-bright field setup for additional light exposure steps.
      NOTE: Before the experiment, measure the power of epi-bright light source and adjust it to the level of own plant growth condition.
4. Run the time-lapse imaging program. For long-term observation over several days, consider watering the plant appropriately, for example, during the light exposure steps.
5. After image acquisition, omit extra channels used for light exposure in the intervals (corresponding to channels 3-8 in Table 1) from the data set. Analyze data with various methods, such as region-of-interest (ROI) analysis, using different image analysis software such as Fiji⁹.