The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).
A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young’s modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.
Plant cells are surrounded by a cell wall that is a complex structure composed of interacting networks of polysaccharides, proteins, metabolites, and water that varies in thickness from 0.1 to several µm depending on the cell type and the phase of growth1,2. Cell wall mechanical properties play an essential role in the growth of plants. Low stiffness values of the cell wall have been proposed as a precondition for cell growth and cell-wall expansion, and there is increasing evidence that all cells sense mechanical forces to perform their functions. However, it is still debated whether changes in the physical properties of the cell wall determines cell fate2,3,4. Because plant cells do not move during development, the final shape of an organ depends on how far and in what direction a cell expands. Thus, Arabidopsis root is a good model to study the impact of cell wall physical properties in cell expansion because different types of expansion occur in different regions of the root. For example, anisotropic expansion is evident in the elongation zone and particularly noticeably in the epidermal cells5.
The method described here was used to characterize the physical properties of the cell wall of epidermal cells at the nanoscale of living Arabidopsis roots using an Atomic Force Microscope (AFM) coupled with an inverted fluorescence phase microscope6. For an extensive revision of the AFM technique, read7,8,9.
This protocol outlines a basic sample preparation method and a general method for AFM-based elasticity measurements of plant cell walls.
Figure 1: Schematic overview of force-indentation experiment in Arabidopsis roots using atomic force microscopy (AFM). The scheme gives an overview of the steps of a Force-Indentation experiment from the preparation of the substrate to immobilize the root sample firmly (1-2), root viability confirmation through propidium iodide staining (3), cantilever positioning on the surface of an elongated epidermal cell of the primary root (4-5), force curves measurement (6), and force curve processing to calculate the apparent Young's modulus (7-8). EZ: elongation zone. Please click here to view a larger version of this figure.
1. Preparation of the plant material and growth conditions
2. Osmotic stress treatment (optional)
NOTE: This section provides details on the growth of Arabidopsis roots in osmotic potential of -1.2 MPa as estimated by cryoscopic osmometer (Table of Materials). This part can be omitted or changed depending on the experimental question at hand.
3. Atomic force microscopy (AFM) nanoindentation experiments
4. Measure the apparent Young's modulus
Force-Indentation experiments
The following text presents some results expected when a force-indentation experiment is conducted to show the typical output to expect when the protocol is well executed.
Force-displacement curves
Representative force indentation plots that were obtained indenting live samples at a position placed in the center of the cell of the root elongation zone are presented in Figure 2. When the AFM tip starts to indent the surface of the cell wall, the force begins to increase (indicated by 1 in Figure 2A) because of the cell wall opposition to the deformation. The increase of force (loading) continues until the maximum force value is reached (indicated by 2 in Figure 2A); after this point, the unloading part of the indentation begins.
Figure 2: Force-displacement curves obtained in indentation experiments made in cell walls of live root elongation zone cells. (A) A typical force-displacement curve. At the position marked by 1, the force begins to increase; the AFM tip starts to indent the cell wall. The increase of force (loading) continues until the maximum force value is reached, as indicated with 2; after this point, the unloading part of the indentation begins. (B) Example of a force-displacement curve in which it is impossible to detect the position of the cell surface before the indentation (indicated by a), and both the loading and unloading curves are noisy. Please click here to view a larger version of this figure.
The expected force-displacement curves will show in the indentation part that the force grows following a parabola, as predicted by the model (explained in step 4.1) (Figure 2A). Another fitting parameter is the contact point position; it 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 (Figure 2B). The loading and unloading curve of the force-indentation experiment must be devoid of noise. Noisy curves such as the one represented in Figure 2B must be discarded.
Normalized histogram of the apparent Young's modulus
The histograms showing the distribution of the frequency of the obtained values of the apparent Young's modulus for a set of 201 successful indentations on nine different cells of three different plants of Col-0 grown in control conditions are presented in Figure 3. The figure shows an example in which the mean and standard deviation of the apparent Young's modulus (88.12 ± 2.79 KPa) were calculated from the histograms fitted with a Gaussian curve6.
Figure 3: Example of a histogram of apparent Young's modulus that allowed a proper statistical analysis of the results. Data were obtained from force curves on nine different cells of three different plants of Col-0 grown in control conditions. The data obtained from 201 Force curves were fitted to a Gaussian distribution. The relative frequency indicates the number of fitted force curves that correspond to each calculated apparent Young's modulus. The mean and standard deviation values of this condition were obtained from the Gaussian fits. RF: Relative frequency; AEM: apparent Young's modulus. This figure was modified from reference6 Please click here to view a larger version of this figure.
Some genotypes could be very difficult to indent due to the morphology of the root. This was the case for prc1-1 mutant and ttl1 prc1-1 double mutant grown in severe osmotic stress. In this condition, these mutants had few elongated cells in the root elongation zone and developed very long root hairs, which interfere with the cantilever producing the sample movement. Figure 4 presents histograms showing the probability distribution of the obtained values of the apparent Young's modulus on nine different cells of the ttl1 prc1-1 double mutant. The figure shows an example in which the obtained force curves did not allow the proper analysis. Only Histogram H that corresponds to one cell could be fitted to a Gaussian distribution6.
Figure 4: Example of histograms of apparent Young's modulus that did not allow a proper analysis. Data were obtained from force curves on nine different cells of three different plants of the ttl1prc1-1 double mutant. The relative frequency indicates the number of fitted force curves that correspond to each calculated apparent Young's modulus. Only histogram H could be fitted to a Gaussian distribution. RF: Relative frequency; AEM: the apparent Young's modulus. This figure was modified from reference6. Please click here to view a larger version of this figure.
Cell and cell-wall mechanics are increasingly becoming relevant to gain insight into how mechanics affects growth processes. As physical forces propagate over considerable distances in solid tissues, the study of changes in the physical properties of the cell wall and how they are sensed, controlled, tuned, and impact the plant's growth are becoming an important field of study2,3,8.
A method is presented here for studying the cell wall physical properties of cells in the elongation zone of the Arabidopsis root. The AFM technique may be readily combined with other techniques, such as growth rate studies, to understand the effect of the cell wall physical properties in root growth.
One of the most critical experimental steps is the mechanical immobilization of the sample; the sample must be firmly attached to avoid the sample movement while it is indented. The layer of the silicone glue must be thin enough to allow it to dry quickly. The manipulation of the silicone glue and the sample should be careful to ensure that the silicone does not cover the areas of the sample that will be measured. Furthermore, the preparation should be completed quickly (within a minute) to maintain the silicone glue's adhesive properties and prevent roots dehydration. It is crucial to prevent tissue damage; dehydration and damage of the epidermal tissue will produce irreversible changes in the mechanical properties of the cell wall. Other methods used immobilization in 1% agarose on a glass slide; however, this method also has the challenge of needing quick manipulation and an additional step of preparation of the glass slides to prevent the samples from moving in the X and Y axes during the analysis. For more information, see references8,18. Another relevant point of this protocol is to count with an optical microscope with epifluorescence coupled to the AFM that has different objectives (10x, 20x, and 40x). This allows selecting the indentation area accurately.
One challenging aspect of the protocol is the selection of the AFM indentations settings. Different indenters and indentation depths render different resolutions and can be used to answer different research questions8. Several studies have shown that the tip's size and geometry are important considerations to discern the desired results. Different information is obtained with different tip shapes7. For example, the conical tip can discriminate mechanical properties at the sub-cellular level, the spherical tip can better represent the mechanical properties of the whole cell19, and pyramidal tips allow to obtain high-resolution images and mechanical properties at the same time7,20. The selection of the pyramidal tip radius (20-60 nm) is important to prevent the tip from sinking, allowing a proper indentation process. For each application, the researcher must choose the type of cantilever (and tip) better suited for the sample and the kind of measurements to perform8.
The method has some limitations: the mechanical properties of only cells at the surface of organs can be measured, and tissues with variable topographies are difficult to work with. For example, root hairs could make it difficult to access the epidermal cells with the AFM tip8,18. Finally, while the method presented measures elasticity, nanoindentation has also been used to report viscosity and turgor pressure in plants and animals8,18.
The authors have nothing to disclose.
This research was funded by CSIC I+D 2018, grant No. 95 (Mariana Sotelo Silveira).; CSIC Grupos (Omar Borsani) and PEDECIBA.
1 x Phosphate-Buffered Saline (PBS) | Include sodium chloride and phosphate buffer and is formulated to prevent osmotic shock and maintain water balance in living cells. | ||
AFM software | Bruker, Billerica, MA, USA | ||
Atomic force microscopy (AFM) | BioScope Catalyst, Bruker, Billerica, MA, USA | ||
Catalyst Probe holder-fluid | Bruker, Billerica, MA, USA | CAT-FCH | A probe holder for the Bioscope Catalyst, designed for fluid operation in contact or Tapping Mode. Also compatible with air operation. |
Cryoscopic osmometer; model OSMOMAT 030 | Gonotech, Berlin, Germany | ||
Murashige & Skoog Medium | Duchess Biochemie | M0221 | Original concentration, (1962) |
Optical inverted microscope coupled to the AFM | Olympus IX81, Miami, FL, USA | ||
PEGAMIL | ANAEROBICOS S.R.L., Buenos Aires, Argentina | 100429 | Neutral, non acidic silicone glue |
Petri dishes | Deltalab | 200201.B | Polystyrene, 55 x 14 mm, radiation sterile. |
Propidium iodide | Sigma | P4170 | For root viability test. |
Silicon nitride probe, DNP-10, cantilever A | Bruker, Billerica, MA, USA | DNP-10/A | For force modulation microscopy in liquid operation. Probe tip radius of 20-60 nm. 175-μm-long triangular cantilever, with a spring constant of 0.35 N/m. |
Tweezers | Sigma | T4537 |