Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.
Plant cell growth is controlled by the mechanical properties of their cell walls. Therefore, it's important to know these properties in different organ and tissues of the plants. The method presented here allows the biomechanical characterization of non-fixed and non-dehydrated cell walls in the internal tissues of young plants.
Start with preparing the solutions and sample for vibratome sectioning. Pour a four millimeter layer of melted 3%agarose on the bottom of the Petri dish and let it cool slightly to prevent thermal damage to the sample. Place about five millimeter long three or four pieces of the plant organ horizontally on the agarose.
Around 30 to 60 seconds later, a thin semi-solid film will appear on top of the first agarose layer. Then, carefully pour a second layer on top. After the agarose is completely solidified, cut out the block containing the specimen.
Shape the block into a hexagonal truncated pyramid to ensure its stability during further sectioning. Before starting the sample sectioning with the vibratome, glue the block to the vibratome stage with cyanoacrylate adhesive. Place the stage in the vibratome, so that one of the pyramid corners faces the blade of the vibratome and pour water into the vibratome bath.
Set the sectioning parameters like section thickness, blade speed, and vibration frequency and cut the sample. Using a fine brush, move the section from the water bath onto a glass slide and place a drop of water on the section to prevent it from drying out. After checking the quality of the section under a light microscope, select the proper section having cell wall perpendicular to the section plane.
Then immobilize the section for atomic force microscopy measurements by pouring a layer of one milliliter of 1%molten agarose on the bottom of the Petri dish cap using a pipette. After the agarose has solidified, remove excess water from the section by bringing filter paper to its edge. Carefully transfer the section from the slide to the center of the Petri dish cap using a brush.
Then carefully add 1%agarose around the section using a 20 microliter pipette and pour the water or other solution for the atomic force microscopy into the Petri dish cap with the immobilized section. Guide the sample under the atomic force microscopy cantilever using the optical microscope. Click on the Approach button and then the Landing button to approach the sample in contact mode with a set point of one nanoampere.
Click on the Scanning button and then the Area button. Select the area size of 50 micrometers by 50 micrometers to scan. Click on the Move Probe button and check the entire scan area by moving the scanner over it and finding the highest point based on the degree of scanner protrusion.
Open the Approach tab. Then click on the Remove button to retract from the sample. Using the highest point as a target, click the Landing button to approach the sample again.
Then check the surface again by clicking on the Move Probe button and moving the scanner over it. Set the scan rate to 0.5 hertz, and set the scan size to 50 micrometers by 50 micrometers and the scan point to 64 by 64. Click on the Run button and scan to check the surface of the sample and it's possible contamination with agarose.
After clicking the On button, choose the HDPlus mode in the dropdown menu in the program's main window and set the set point to 0.1 nanoamperes in the main program window. In the main tab of the HD window, set the scanning parameters appropriate to the studied sample. Then open the Noises tab in the HD window and enter the cantilever's resonance frequency.
Open the Quant tab of the HD window and enter the IOS, cantilever stiffness, tip radius, and angle. Select the model of contact, which will be used for calculations depending on tip geometry. After this, open the Scan tab of the HD window and select the signals, as well as the direction in which the signal is recorded.
Tick the Force Volume box to get a record of all force curves and click on the Off button at the top of the main program window to switch on the feedback loop Click on the Phase Core button in the main HD window to correct the sensitivity of the optical system. The Versus Time tab of the main HD window presents the function of the DFL signal versus time in real time. Select the parts of this function that will be used for baseline level determination and to fit the contact model for further calculations.
Now, set the scan point value to 256 by 256 in the main window of the program. Then, set the scan rate to 0.2 hertz and click on the Run button to scan the sample. After scanning stops, click on the On button at the top of the main window to switch off the feedback loop.
Choose Contact Mode in the dropdown menu, open the Approach tab, and click on the Remove button to retract from the sample. Click on the Data button to open the analysis software and save the output. Open the saved file in the analysis software.
Select the HD force volume frame. Hold down the Control key and select one visual frame obtained in the same scanning direction. Click on the Load External Map button to see where the cell walls are located.
Check InvOptSens and cantilever stiffness values in the main tab. Open the Additional tab and check the tip parameters and contact model. Click on various points on the cell walls on the visual frame and select only those curves well-described by the model.
The white areas in the modulus map correspond to an erroneous overestimation of Young's modulus due to the scanner reaching its limit in the Z direction. This image is not convenient to be used as an external map for a further selection of satisfactory force curves. However, the DFL signal map presented on the right is better suited here.
Different instruments may refer to DFL signals as deflection or error signals. The scanner that has been fully extended while trying to reach the bottom of the sample can result in erroneous measurements and even interrupted scans. The agarose presence can be checked while obtaining the first scan in the contact mode as the cell walls and some cell bottoms are visible on such scans.
However, in case of inaccurate immobilization, the surface may be covered with agarose, which masks the sample topography. Out of four different force curves recorded at different points of the same cell wall, the curve recorded at 0.1 shows no baseline, meaning no separation of the cantilever tip from the cell wall. The curve recorded at 0.3 shows a shoulder on the approaching part indicating bending of the cell wall.
Points two and four show satisfactory force curves with similar moduli values. Sample preparation and examination require practice, but it can be mastered. A crucial part of this protocol is to filter resulting curves.
Do not rely on moduli, which were calculated automatically. The same technique can be used to measure the adhesion forces or energy dissipation. It also can be important for description of viscose or viscoelastic behavior of some materials.
