This method allows to quantify changes in the physical properties of the plant cell wall during development and relate this microscopic changes to the growth of an entire organ. The main advantage of this technique is that it's not invasive and it does not require any treatment that allows the in vivo quantification of physical properties of the cell wall add subcellular resolution relatively rapid. To begin, spread a thin layer of silicone glue into the Petri dish with a cover slip and leave it in the air for 45 seconds.
Using tweezers, place the seedling on the glue and orient its direction to avoid contact between the seedling's protruding parts, and the cantilever. Then press the root gently to the silicone glue layer to bind it firmly. Leave it for 45 seconds, and add the 1X PBS solution.
Mount the standard silicon nitride cantilever with a pyramidal tip into the AFM probe holder for fluid, and align the laser on the cantilever close to the position of the tip. Then move the photo diode to place the laser spot at the center of the detector. Calibrate the deflection sensitivity by performing an indentation with a ramp size of 3 micrometers, an indentation and retraction rate of 0.6 micrometers per second and a trigger threshold of 0.5 volts.
Ensure that the probe is not interacting with the sample and calibrate the cantilever's spring constant. Using the thermal tune utility, click on calibrate followed by thermal tune or on the thermal tune icon in the nano scope toolbar and enter the cantilever temperature. After selecting a frequency range, click on the sample harmonic oscillator fluid button.
Next, click on acquired data in the thermal tune panel. Now, adjust median filter width to three. Adjust PSD binwidth to reduce the noise in the acquired data by averaging and set fit boundaries around the first resonance peak.
Click on calculate spring constant K, and then click on yes in the pop-up window, asking if the user wants to use this value. Using the inverted optical microscope at 10, 20 and 40 times eye piece magnification, position the AFM probe on the surface of the fourth elongated epidermal cell of the primary root ensuring to position it in the center of the cell. With the previously calculated spring constant value obtained force curves with a ramp size of 3 micrometers, a trigger threshold of 11 nanonewton, and an indentation and retraction rate of 0.6 micrometers per second at selected points.
Obtain force curves from three cells per root for each treatment and capture at least 150 force curves for each root. This plot shows an expected result when a force indentation experiment is conducted on live samples positioned at the center of the cell of the root elongation zone. When the AFM tip starts to indent, the surface of the cell wall, the force begins to increase because of the cell wall opposition to the defamation.
The increase of force continues until the maximum force value is reached. After this point, the unloading part of the indentation begins. The force grows following a parabola in the indentation part, which is important to fit each curve to the prediction model for pyramidal indenters used for calculations.
As a fitting parameter, the contact point position should correspond to the cell wall surface before indentation and is considered the origin of the AFM tip displacement. Force curves in which it is impossible to detect the point of contact before the indentation should be discarded. Further, the loading and unloading curve of the force indentation experiment must be devoid of noise.
After fitting each force curve to the model, histograms were obtained that showed the distribution of the frequency of the obtained values of the apparent Young's modulus. This histogram shows the frequency obtained with a set of 201 successful indentations on nine different cells of three different plants of Columbia Zero grown in control conditions. Indentation could be difficult for some genotypes due to the morphology of the root.
For example, the TTL 1 PRC 1-1 double mutant grown in severe osmotic stress. These histograms show the probability distribution of the obtained values of the apparent Young's modulus from nine different cells. Only histogram H that corresponds to one cell could be fitted to a Gaussian distribution.
The metal presented here could be combined with other techniques such as grow rate studies or analysis of the chemical composition of the cell wall. These other techniques will further complement information to understand how the chemical composition on the physical properties of the cell wall affect the growth of an organ.
