1.Measure of Cell Wall Mechanical Properties
NOTE: Example of the developing gynoecium of Arabidopsis is presented.
2. Measure of Turgor Pressure
NOTE: An example of the oryzalin-treated inflorescence meristem of Arabidopsis is presented.
Figure 1A and Figure 1B show a screenshot illustrating the result of the steps 1.3.4 to 1.3.6 of the protocol, used to locate a region of interest where to acquire the QI map. It is worth mentioning that the region of interest has been chosen in order not to be on a tilted surface (i.e., as flat as possible). Actually, as noticed by Routier et al.5, if the indentation axis is not perpendicular to the surface, the Young's modulus measured can be underestimated. This effect is visible on the upper-right corner of C or D panels of Figure 1, where part of the cells' border looks softer than the rest of the cell. The artefacts induced by this effect will be far stronger in the case of meristems, where the local tilt angle can easily overcome 40° over 50 x 50 µm2 scan areas. In our laboratory, we are currently developing an algorithm, based on the approach described by Routier et al.5, to correct those artefacts (paper in preparation). Figure 1C and Figure 1D show Young's modulus maps obtained after the analysis detailed in the fourth paragraph of the protocol. In particular, panel C represents the Young's modulus obtained analyzing the whole indentation, up to the user-defined force setpoint, while panel D shows the result of the analysis of the first 100 nm of indentation (as described in step 1.4.9). Here, the 2 maps look highly similar, because during the experiment set-up, the setpoint has been chosen in order to obtain average indentations around 100 nm. The variation of the analyzed indentation can sometimes lead to better highlight sample heterogeneities, that can be helpful for identifying the location or for providing information on the behavior of internal structures (e.g., Costa et al.10)
The force curve in Figure 2 shows two effects that are worth mentioning. First of all, it can be noticed that if the approach part of the force curve actually ends at the setpoint force of 500 nN, the downward tip movement keeps on going, meaning the final force applied by the tip is higher than expected (here 1,000 nN). This is due to the fact that the feedback loop monitoring the cantilever deflection does not act instantaneously, so its finite reaction time introduces a lag between deflection threshold detection and piezo movement stop. Furthermore, especially when using CellHesion which moves the sample holder, inertia of the moving parts comes into play, making this time lag longer. This issue can be limited by using the head piezo, in which case the user must be really careful in the case of measurements on very rough or tilted samples, or by reducing the ramp velocity, which anyway limits the resolution and/or the number of samples scanned (furthermore for too slow maps, the sample growing can become an issue). Generally, if the force curves are far enough from one another (based on the indentation model of choice, the radius of the indented area can be calculated given the indentation depth and used as minimum separation distance), the measurements performed in different positions along the sample should not be correlated and if the analysis is performed on the approach curve, overcoming the force threshold should not be an issue. Anyway, if the sample is delicate or if repeated scans are done on the same area, the scientist may want to try to minimize this overshoot. Unfortunately, there is not a rule of thumb to determine the amount of this overshoot, since it depends on the piezo used, on the ramp velocities, but also on material properties: the stiffer the material, the faster the variation of the deflection signal in time and, given a finite feedback response time, the higher the overshoot.
The second thing to notice is the waving on retract curve highlighted by the ellipse. Such waving can be an indicator of sample moving/vibrating under the action of the tip. In this case, the user can try to change the scanned area (sometimes other part of the microscope but the tip can touch the sample, or the sample can be insufficiently well fixed to its support) or the sample if several of them have been prepared on the same support. If the feature is always there, it is better to change the fixation method, in this case switching from the double-sided tape fixation to biocompatible glues.
Figure 3 depicts the determination of key parameters for turgor pressure deduction. Every parameter, except for cell wall thickness11 which was separately determined by electron microscopy to be ~740 nm, can be retrieved from AFM scans and indentations. Following the fitting process in step 2.4.10, the turgor pressure is deduced to be 1.50 MPa for this force curve. Pooling all eighteen force curves in this cell yields average values of E = 6.77 ± 1.02 MPa, k = 14.18 ± 2.45 N/m and P = 1.45 ± 0.29 MPa (mean ± standard deviation).
Figure 1: Topography maps (Height Measured) typically acquired during an experiment. (A) A first low resolution/low force map is recorded to find a suitable region to scan. (B) A smaller high resolution/higher force map is acquired for the calculation of Young's modulus (on the area highlighted by the black dotted square). (C) Young's modulus maps calculated from B map, as detailed in step 1.4. Here, the Young's modulus map has been obtained analyzing the whole indentation on each force curve. (D) Young's modulus map obtained analyzing only the first 100 nm of indentation. Please click here to view a larger version of this figure.
Figure 2: Example of force curve. In blue the approach, in red the retract segment. Force maximum (located on retract curve) is different from the force setpoint because of finite feedback loop reaction time and because of inertia. The waving in retract curve can be representative of a deficiency in sample fixation to its support. Please click here to view a larger version of this figure.
Figure 3: AFM measurements for turgor pressure deduction. (A) DMT modulus map shows clear cell contour to supervise the positioning of deep-indentation sites near cell barycenter. Indentation sites are marked by crosses. (B) Force curve of deep indentation at the red cross position in A. Cell wall Young's modulus E (green) and sample apparent stiffness k (red) are fitted at different regimes of the curve as detailed in step 2.4. R2 denotes coefficient of determination of the fits. (C) Height map with manually determined long and short axes of a, depicted by white lines, of the same cell analyzed in panels A and B. (D) Height profile of the long and short axes of the cell in Panel C (blue, long axis red, short axis) and the fitted radii of curvature (r1 and r2). Please click here to view a larger version of this figure.
Growth medium | |||
1000x vimatin stock solution | used to make ACM, composition see Stanislas et al., 2017. Add to ACM after autoclaving, before pouring. | ||
1-N-Naphthylphthalamic acid (NPA) | Sigma-Aldrich/Merck | 132-66-1 | add to Arabidopsis medium, 10 μM. Add after autoclaving, before pouring. |
agar-agar | Sigma-Aldrich/Merck | 9002-18-0 | add to Arabidopsis medium, 1% w/v. |
agarose | Merck Millipore | 9012-36-6 | used to make solid ACM, 0.8% w/v. |
Arabidopsis medium | Duchefa Biochimie | DU0742.0025 | For in vitro arabidopsis culture, 11.82g/L. |
Calcium nitrate tetrahydrate | Sigma-Aldrich/Merck | 13477-34-4 | add to Arabidopsis medium, 2mM. |
MURASHIGE & SKOOG MEDIUM | Duchefa Biochimie | M0221.0025 | Basal salt mixture, used to make ACM, 2.2g/L. |
N6-benzyladenine (BAP) | Sigma-Aldrich/Merck | 1214-39-7 | used to make ACM, 555 nM. Add to ACM after autoclaving, before pouring. |
oryzalin | Sigma-Aldrich/Merck | 19044-88-3 | for oryzalin treatement, 10 μg/mL. |
plant preservation mixture (PPM) | Plant Cell Technology | used to make ACM, 0.1% v/v. Add to ACM after autoclaving, before pouring. | |
Potassium hydroxide | Duchefa Biochimie | 1310-58-3 | used to make Arabidopsis medium and ACM, both pH 5.8. |
sucrose | Duchefa Biochimie | 57-50-1 | used to make ACM, 1% w/v. |
Tools for AFM | |||
BioScope Catalyst BioAFM | Bruker | The AFM used for turgor pressure measurement in this protocol. | |
Nanowizard III + CellHesion | JPK (Bruker) | The AFM used for measuring mechanical properties. | |
Patafix | UHU | D1620 | |
Reference elasitic structure | NanoIdea | 2Z00026 | |
Reprorubber-Thin Pour | Flexbar | 16135 | biocompatible glue. |
Spherical AFM tips | Nanoandmore | SD-SPHERE-NCH-S-10 | Tips used for measuring mechanical properties. |
We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.
We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.
We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.