Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

1.Measure of Cell Wall Mechanical Properties

NOTE: Example of the developing gynoecium of Arabidopsis is presented.

1. Preparation of the biological samples
   1. Collect a closed flower bud at stage 9 to 10 (about 0.5 mm long) according to published stages determination for Arabidopsis⁴. Under a binocular, using fine tweezers, carefully open the bud to check the stage of development and collect the gynoecium located at the center of the flower.
   2. Put the gynoecium on double side tape placed at the center of the cover of a small Petri dish (diameter of 5 cm).
      NOTE: As an alternative, biocompatible glue can also be used for more efficient immobilization of the sample upon indentation.
   3. Rapidly add water until the sample is completely covered. This avoids dehydration and reduces adhesion of the tip to the sample. Alternatively, submerge the sample in liquid medium, such as Arabidopsis apex culture medium⁵.
2. AFM calibration
   1. Set the cantilever spring constant k high enough to allow the deformation of the sample surface up to the desired indentation, but not too high to avoid loss of sensitivity.
      NOTE: As a rough rule of thumb, if Young's modulus of the sample is known, the order of magnitude of the spring constant can be chosen as k≈E * δ, where δ is the desired indentation.
   2. Use an R = 400 nm spherical-ended tip with 15 µm tip-cantilever distance.
      NOTE: The tip radius is directly related to lateral resolution. Generally, for indentation on biological materials, choose rounded tips (R greater than 10-20 nm) or colloidal probes. Small colloidal probes may be tricky to use due to the small distance between tip end and cantilever that can touch sample surface.
   3. Switch on the software and place the head horizontally at least 2-3 h before the experiment: this will allow the head to thermalize and will avoid thermally-induced cantilever-laser relative movements. If the microscope is equipped with a CellHesion module (extending the available Z piezo range to 100 µm instead of 15 µm), switch on its controller first and then select CellHesion mode when starting the software.
   4. Mount the cantilever on the glass block and mount the block on the head. Place a droplet of a few microliters of ultrapure water on the tip to avoid the formation of air bubbles when the tip is dipped into water.
   5. Place a hard sample (cleaned glass slide or sapphire) and add 30-50 µL of ultrapure water.
      NOTE: The calibration procedure described here is the sometimes-called contact calibration. First, a force curve is made on a stiff and flat surface and then the oscillation spectrum of the thermally excited cantilever is recorded in order to calculate the spring constant. Other calibration protocols exist and will briefly be described in the discussion paragraph.
   6. Place the head on the stage (be careful to lift the Z motors high enough). Use the optical image to roughly place the laser on the cantilever.
      1. Move the laser along the main axis of the cantilever monitoring the sum signal on the photodiode.
         NOTE: When standard cantilevers are used, a sum greater than 0.5 V should be obtained.
      2. Move the laser along the other direction and maximize the sum signal in order to position the laser at the middle of the cantilever. This will minimize the cross-talk between lateral and vertical deflection.
   7. Measurement of the deflection sensitivity
      NOTE: The photodiode reads laser displacement and provides a signal in volts. In order to be able to measure deflection in metric unit, deflection sensitivity must be measured.
      1. Set the instrument in Contact → Force spectroscopy. Set a Relative setpoint to 2 V, Z length to 0.5 µm, and Extend speed to 2 µm/s (Sample rate to 10000 Hz) and select Z Closed loop.
      2. Open the Calibration manager and select the contact part of the force curve (that should be linear) to make a linear fit: the inverse of the slope gives the deflection sensitivity. The photodiode reading is now calibrated in metric unit.
   8. Determination of spring constant. In Calibration manager, select Spring constant to run a thermal spectrum acquisition. To average the signal for a longer time, select the ∞ symbol. The power spectrum of the thermally excited cantilever may show several peaks; draw a selection around the one placed at lowest frequency to fit it.
      NOTE: If thermal tune is performed in liquid, the resonance peak will be broader and its frequency lowered compared to nominal one.
3. Force spectroscopy experiment set-up and acquisition
   1. Place the sample on the AFM stage and place the head over the sample.
      NOTE: Be sure the head has been retracted enough to avoid a hard contact between the tip and the sample surface.
   2. Let the cantilever thermalize for a few minutes.
   3. In QI mode, approach with a Setpoint force of 50 nN.
   4. Set a Z length of 4 µm and a scan area to 80 x 80 µm² with a number of pixels of 40 x 40. In the Advanced imaging settings panel, set the mode to constant speed. Set extend and retract speed to 200 µm/s and Sample rates to 25 kHz.
   5. Start scanning and use this rapid low-force scanning for checking if the sample moves. Verify that the scanned area is free from debris or deflected cells and locate a region of interest as flat as possible to perform the measurements.
      NOTE: In order to appreciate the real sample tilt, the line levelling should be set to OFF or to Constant. An excessive tilt angle between the indentation axis and the surface will have an effect on the measured Young's modulus⁵.
   6. Once an area of interest has been located, select a region of 40 x 40 to 60 x 60 µm² around it and increase the pixel number to reach 2 pixels/µm. Increase the Setpoint to 500 nm to obtain 100-200 nm of indentation. Adjust this value, if necessary, at the beginning of the experiment. Decrease the Z length to 2 µm. Decrease extend and retract speed to 100 µm/s and increase the Sample rate to 50 kHz.
   7. Start scanning and save the output (generally composed by an Image and a Data file).
   8. At the end of the measurement day, remove the tip holder and rinse it gently with ultrapure water and 70% EtOH.
   9. Dry and remove the cantilever. For further experiments with the same tip, consider cleaning it with a wet-cleaning protocol and, if possible, a follow-up plasma O₂ treatment. Do not let the water dry on the cantilever and/or tip holder to avoid salt crystallization.
4. Data Analysis (for 6.x Data Processing software version)
   1. Open Data Processing software and load the Data file.
   2. Click on Use this map for batch processing button in order to use the same analysis parameters on all the curves of the map.
   3. In Load pre-defined process, select Hertz fit.
   4. Use the first tab to verify or change the calibration parameters.
   5. In the second tab, remove an offset (or an offset plus a tilt) from the baseline to set its average value to 0.
   6. In the third tab, estimate the point of contact (POC) position by considering it as the first point crossing the 0 force when coming down from the setpoint value along the extend curve.
   7. The tab Vertical tip position calculates tip movement by subtracting the cantilever deflection from Height Measured. At this step, use Unsmoothed height (raw data) for the following fit, by checking the corresponding checkbox.
   8. In Elasticity fit tab, select the appropriate fit model. If no or weak adhesion is visible on retract curves (corresponding to less than 10% of the setpoint force or to the maximum average force at the selected indentation depth), the Model type should be set to Hertz/Sneddon and extend curve should be used. In case of stronger adhesion, DMT model, which stands for the Derjaguin-Muller-Toporov model, should be preferred and fit should be performed on retract curves (refer to manual for details on the available contact models and related formulae).
      1. Set tip geometrical parameters based on nominal tip shape. Here, Tip Shape is sphere and Tip Radius is 400 nm.
      2. Set the Poisson's Ratio to 0.5 as it is conventionally done for biological material (corresponding to incompressible material).
   9. Fit up to specific indentation. By default, the fit is performed over the whole curve. If the fit has to be done up to a specific indentation, first check the Shift Curve checkbox; this will shift the origin of the curve based on newly determined baseline and POC values.
      1. Add a second Elasticity fit routine by clicking on the icon on the main window.
      2. Set again all the fit parameters and specify in X min the desired indentation. Add as many steps as necessary (2 to 3 steps should be enough) in order to refine the determination of the POC position and subsequently the calculated indentation depth. The process can be saved at this point.
   10. Click on Keep and apply to all to iterate the previous steps on all the curves of the map.
   11. Save the results. An image and a .tsv files will be obtained.

2. Measure of Turgor Pressure

NOTE: An example of the oryzalin-treated inflorescence meristem of Arabidopsis is presented.

1. Preparation of biological sample
   1. Collect Arabidopsis inflorescence meristem (IM) treated with the microtubule depolymerizing drug oryzalin from in vitro plantlet grown on medium containing the polar auxin transport inhibitor 1-N-Naphthylphthalamic acid (NPA) following published method⁶.
   2. Mounting IM sample following one of the two options
      1. For long-term monitoring: mount the sample in a Petri-dish containing Arabidopsis apex culture medium (ACM)^7,8 and 0.1% plant preservation mixture (PPM), to prevent contamination. Suspend the IM tip above ACM surface and support the base of IM with a drop of 2% agarose.
      2. For measurement with rapid solution changes:mount the sample in a Petri-dish holding a small piece of adhesive mastic, and quickly seal the gap between the mastic and the sample base with bio-compatible glue. Wait for the glue to solidify (less than 2 min), then submerge the sample in liquid ACM containing 0.1% PPM.
         NOTE: Make sure the sample surface is not coated with agarose or glue when fixing the sample base. AFM measurement of turgor pressure requires the sample to be stably mounted, and the previous mounting methods provide acceptable stability. Depending on the sample, other mounting methods might be used, like double-sided tape, poly-lysine, etc.
2. AFM calibration
   1. Turn on the AFM acquisition software and choose the PeakForce QNM (large amplitude) measurement mode.
   2. Perform calibration following the same principle described in steps 2.1 to 2.6.
   3. In the Check parameters window, set Scan size to 0. In the Ramp window, set Ramp size between 200 nm and 500 nm, Trig threshold between 2 V and 5 V and Number of samples to 2048 or higher.
   4. Align the cantilever tip with the calibration sample and click Approach.
   5. Upon contact, go to Ramp window and click the button Continuous ramp. On the curve's linear regime, determine the slope by clicking on the Update Sensitivity button. Repeat the deflection sensitivity measurement several times and manually update the calibration with the measurement average by opening the Tab Detector from the menu Calibration.
   6. Retract the AFM head and remove the calibration sample.
      NOTE: It is suggested to completely retract AFM head by choosing tab Stage → Initialize to prevent accidental hard contact upon sample change.
3. Force spectroscopy experiment set-up and acquisition
   1. If the sample is not submerged yet, submerge it with liquid ACM containing PPM.
   2. In the acquisition software, specify measurement parameters as follows:
      1. In Check parameter window, set Spring constant to the cantilever's manufactured spring constant or the determined spring constant as in step 2.8. In this example, it is set at 42 N/m.
      2. Set Tip radius to 400 nm in this example.
      3. Set Sample Poisson's ratio to 0.5, since water contributes mainly to turgor pressure.
      4. Set Sample/Line to 128 to ensure rapid acquisition.
      5. Set Scan rate to 0.2 Hz.
      6. Set Scan size to 1 µm.
         NOTE: Setting scan rate and scan size small may effectively prevent AFM scan-triggered sample damage. It is recommended reducing these two parameters upon any sample change.
      7. In Ramp window, set Ramp size to 5 µm.
         NOTE: It is better setting ramp size bigger than intended indentation depth for better baseline acquisition.
      8. Set Trig threshold to maximum.
      9. Set Number of samples to 4608.
      10. Optionally, in Microscope → Engagement parameters tab, reduce the following parameters to prevent strong contact-induced sample damage. Set Peak Force Engage Setpoint to 0.3 V (default 0.5 V). Set Engage int. gain to 0.5 (default 0.75). Set SPM engage step to 4 µm (default 15 µm).
   3. Place and align the sample under the AFM head, and approach until cantilever is submerged but not in contact with the sample surface.
      NOTE: While approaching, lightly blow on the surface of liquid ACM until a liquid bridge is formed between the cantilever and the liquid surface. This usually prevents hard contact.
   4. With care, manually approach towards the sample. When the probe is relatively close to sample surface, click Approach.
   5. Upon contact, gradually increase Scan size and/or Scan rate until a desired balance without damaging the sample and/or the cantilever.
      NOTE: The scan size is limited by the surface curvature and roughness of the sample. In the case of oryzalin-treated IM, 50 x 50 µm² scan area can be achieved with scan rate of 0.3 Hz.
   6. While scanning, determine if the measurement region is as desired. Relocate if needed. When satisfied, click the button Point and Shoot to initiate the point and shoot window.
   7. Before recording the scan, choose an appropriate image channel that may facilitate clear identification of cell contours. Often, Peak force error, DMT Modulus, LogDMT Modulus or Dissipation is suitable. Specify save directory and file name. Then click Ramp on next scan to initiate recording.
      NOTE: A string of a dot and three numbers will be automatically added after your designated file name (like .000). This number automatically increases by 1 upon each saved scan repetition.
   8. When scan is complete, the software interface will be automatically redirected to Ramp window. Click on the scanned image to specify the positions to indent.
      NOTE: Before recording indentations, it is better choosing several landmarks to perform test indentations by clicking Ramp only, in case parameter alternation is required (for Ramp size, Piezo position, etc.). Indentation depth needs to be bigger than the cell wall thickness (ideally determined separately by transmission electron microscopy).
   9. Choose at least three indentation sites per cell near its barycenter, and repeat indentation three times per site. This would yield at least nine force curves per cell for further analysis. When satisfied with indentation site placement, click Ramp and capture. The force-indentation curves are automatically saved to the designated directory.
   10. When the ramps are completed, relocate to a different position for tile measurement, or retract scan head and change sample.
   11. When finished, clean the cantilever as in steps 1.3.8 and 1.3.9.
4. Data analysis
   1. In the analysis software, open the *.mca file. This shows the position of each force curve on the scanned image. If desired, pre-select force curves for analysis.
   2. Open one force curve to be analyzed, usually in the format of x0000y.00z, where x is the specified file name in the save directory while y and z are automatically registered numbers denoting indentation sequence and scan number.
   3. Click the Baseline correction button and drag the blue dash lines on the force curve until Extend Source Baseline Start and Extend Source Baseline Stop are at 0% and 80%, respectively. Click Execute.
      NOTE: Alternatively, Extend Source Baseline Stop may be set at different values, as long as it is still within the baseline and not beyond the contact point.
   4. Click the Boxcar Filter button and click Execute to smooth force curve.
   5. Click the Indentation button.
      1. In the Input window, set the Active Curve 세스 Extend.
      2. Set the Fit Method 세스 Linearized Model and Include Adhesion Force 세스 Yes.
      3. Set Max Force Fit Boundary to 99% and Min Force Fit Boundary to 75%.
      4. Set Fit Model 세스 Stiffness (Linear).
         NOTE: This setup will compute the apparent stiffness k for turgor pressure deduction. In this example, the fit boundaries reflect a stiffness fit of around 1.5 µm indentation depth.
   6. Force curves can be analyzed in batch. Click the Run History button, specify report directory and add all other force curves that require the same treatment. When satisfied, click Run. By default, the fit will be stored as a *.txt file.
   7. When k is batch fitted, click History → 5 Indentation to return to the indentation window.
      1. Change Max Force Fit Boundary to 10% and Min Force Fit Boundary to 0%.
      2. Set Fit Model 세스 Hertzian (Spherical).
         NOTE: This will compute the cell wall Young's modulus E for turgor pressure deduction. In this example, the fit boundaries reflect a Hertzian fit (using spherical probe) of around 0.4 µm indentation depth.
   8. Repeat step 2.4.6 for batch fit for E.
   9. Open the *.00z file (z is the automatically registered scan number) to display the different scan channels. In Height channel window, click Section button. This will allow the measurement of sample's surface curvature that is required for turgor pressure deduction.
      1. Draw a line across the long axis of one cell, move the dash line boundaries to the cell edges, and record the Radius value r₁. Repeat this for the short axis to retrieve radius r₂. Calculate cell's surface mean curvature κ_M and Gaussian curvature κ_G using the two radii measurement as follows:
          and  
   10. Deduce turgor pressure P using the thin-shell model published⁹ as follows:
        
       with
       
       where t is cell wall thickness determined by e.g. electron microscopy.
       NOTE: The deduction of turgor pressure is a fitting process, where iterations are required. Four iterations are generally able to reproduce stable product, however more iterations can be done (for example 100 times).
   11. Calculate mean E, k and P per cell. Also, register the intracellular variability (e.g., standard deviation) for documentation.