An atomic force microscopy based nanoindentation method was employed to study how the structure of cellulose microfibril packing and matrix polymers affect elastic modulus of fully hydrated primary plant cell walls. The isolated, single-layered abaxial epidermis cell wall of an onion bulb was used as a test system since the cellulose microfibril packing in this cell wall is known to vary systematically from inside to outside scales and the most abundant matrix polymer, pectin, can easily be altered through simple chemical treatments such as ethylenediaminetetraacetic acid and calcium ions. Experimental results showed that the pectin network variation has significant impacts on the cell wall modulus, and not the cellulose microfibril packing.

Plant cell walls serve a variety of important biological functions for plants, such as providing mechanical support and determining cell size and shape.1,2 They have also been an interesting subject for engineering applications. Plant cell walls are essential in various commercial products such as paper, textile, plastic, etc. More recently, they have gained intensive attention as potential resources for biofuel.3–6 Also, the sophisticated composite structure, naturally occurring in cell walls, inspires innovative designs of engineering materials.7–10 All these applications require understanding of the composite structure of cell walls and their mechanical properties.

Primary cell walls are formed during the growth and division of plant cells. They provide mechanical support for the cells and can expand to allow cells to grow.2,11 Primary plant cell walls are polysaccharide-rich complex structures. The wall contains three main components: cellulose, hemicelluloses, and pectin.12 Cellulose microfibrils are embedded in a highly structured polysaccharide matrix, which consists of hemicelluloses and pectin.13–15 Cellulose is comprised of many parallel chains of β-1,4-glucan with high degree of hydrogen bonding networks, which make cellulose crystalline, strong, and indigestible by humans.16 Cellulose microfibrils serve as reinforcement material of cell wall composite. Cellulose microfibrils are several nanometers in diameter and have varied lengths.17 Hemicellulose is also a polysaccharide, but it is typically made up of chains of xylose interspersed with side chains containing arabinose, galactose, mannose, glucose, acetyl, and other sugar groups, depending on the plant type.18–20 Hemicelluloses fill the space between cellulose microfibrils.21 Pectin is a gel phase that embeds the cellulose-hemicelluloses network.21 Pectin includes relatively simple polysaccharides and becomes soluble with simple treatments such as boiling water or mildly acidic solutions.2 

The analysis of micro- or nano-mechanical properties of plant cell walls has become increasingly important in understanding cell wall structure and cell growth. Indentation technique can investigate the mechanical properties of thin, small, and heterogeneous materials. Nanoindentation has been used to measure hardness and Young's moduli of spruce cell-wall, bamboo cell walls, individual wood fibers, and crop stalks cell walls.22–25 These experiments were done with add-on force transducers or special indenter testing systems. However, in traditional nanoindentation experiments, samples were dehydrated and many were embedded in epoxy resin. Such sample preparations may cause undesired modifications and influence the mechanical properties of the cell walls. In addition, the indent force is in a range of 1–100 μN and the indentation depth is typically comparable to or larger than the cell wall thickness.18,19

With its pico-newton force sensitivity and nanometer displacement accuracy, the atomic force microscopy (AFM) has been recognized as a useful tool measuring the elastic moduli of biological samples.26–30 The nano-indentation with an AFM has been recently applied to living plant cells in conditions close to natural. Mechanical properties of suspended grapevine cells cultured in liquid medium, the primary cell wall of shoot apical meristems, rosette leaves, tomato cells, and epidermal cells of living roots of Arabidopsis thaliana were measured using AFM based nanoindentation.30–34 The indentation depth is much smaller (about 100 nm) than the conventional nanoindentation. However, the turgor pressure of live cells affected the measured results when the whole cell was tested. Routier-Kierzkowska et al. showed that the local stiffness of onion cells increases with larger turgor pressures.35 Forouzesh et al. measured the moduli of plasmolyzed, normal, and turgid Arabidopsis cells. They found that the moduli were lower for the turgid sample even though it has the higher turgor pressure than the plasmolyzed and normal samples.36 Thus, no conclusive understanding of the cell mechanical properties could be deduced.

The aim of this work is to measure the elastic properties of cell walls isolated from onion scales using AFM based nanoindentation. In order to avoid artifacts due to the turgor pressure, a freshly peeled single-layer onion abaxial epidermis wall was fixed on a slide glass and tested in buffer solutions. The elastic modulus of the cell walls in aqueous solution was determined by analyzing the force-distance curve with the Hertzian contact model. In our previous work,34 the cellulose microfibril orientation of the abaxial epidermal walls was found to vary gradually from the dispersed arrangement in the inner scales to the transverse orientation in the outer scales.37 Based on this observation, an interesting hypothesis could be made: the mechanical property of cell wall might gradually change with the variation of cellulose microfibrils orientation and alignment. This hypothesis assumes that the cellulose microfibril packing and orientation may play important roles in the cell wall mechanical properties. Alternatively, it is also possible that the matrix component such as pectin may have strong impacts on the elastic modulus of cell walls. The latter hypothesis could be tested by chemical treatments that modify pectin networks. Quantitative comparison of the nanoindentation measurement results with build-in modulus calculation function of PeakForce QNM® (Quantitative NanoMechanics) mode was made to examine the accuracy of the commercial software.

The experiments in this work were conducted with onion (Allium cepa L. cometa) bulbs obtained from a local grocery. Abaxial epidermal layers were carefully peeled and prepared for experiments. The onion scale was hand-sectioned tangentially under a dissecting microscope (Olympus). Sections were then stained with 0.1% toluidine blue for 1 min at room temperature on a slide followed by rinsing with deionized water and imaged with an optical microscope (EVOS, AMG). The never-dried abaxial epidermal cell wall thickness of the sample onions was determined with optical microscopy.

Onion epidermis cell wall samples were prepared for AFM imaging as described by Zhang et al.38 Freshly peeled cell walls were fixed onto glass slides with edges glued. The epidermal strips were then immersed in a phosphate buffered saline solution (pH 7.4) containing 0.1% Tween-20 for 1 h. After that, the samples were rinsed with distilled water. The inner side of the epidermal wall strips was probed by AFM (Veeco Dimension ICON from Bruker) in PeakForce QNM fluid mode. Nanoscope (v 8.10b44) was used to control AFM operation and Nanoscope Analysis (v 1.40) was employed for image processing. A cantilever holder from Bruker for liquid imaging was used to keep samples fully hydrated in water during the experiments. The tips used for images and indentation are ScanAsyst-Fluid + probes from Bruker with a nominal spring constant given as 0.7 N/m. The spring constant of each tip was calibrated using a thermal tuning method before experiments.39 All images were generated with scanning in the direction along the longitudinal direction of the cells. Figure 1 shows a top view of AFM tip engaged in epidermis cell walls. The force-distance (F-D) curves were generated in a ramp mode with controlled maximum force (15 nN) and constant tip velocity (800 nm/s). The curve generating positions are in the middle of the cells, thus avoiding area with vertical adjacent walls.

FIG. 1.

A typical experimental view during imaging and nano-indentation of hydrated onion cell wall (2nd scale) with AFM.

FIG. 1.

A typical experimental view during imaging and nano-indentation of hydrated onion cell wall (2nd scale) with AFM.

Close modal

The mechanical property measurement with AFM nanoindentation is based on force-distance (F-D) curve analysis. An F-D curve is a plot of tip-to-sample force versus the extension of the piezoelectric scanner measured with a position-sensitive photo detector. The F-D curve provides information of important mechanical properties such as adhesion, impurities, viscosity, and local variations in the elastic properties of the surface.40–43 Figure 2 shows a representative F-D curve obtained on onion epidermis cell wall in liquid. F-D curves can vary with different samples. In our experiments, because the cell wall polymers are hydrophilic (except cuticles in the outside layer), the adhesion between cell walls and clean SiO2 tip surface is very small in water. Thus, a simple Hertz model can be used, especially when the applied load is much larger than the adhesion force.

FIG. 2.

A representative F-D curve (approach and retract) obtained in the experiment on onion epidermis cell wall in liquid. The y axis is the recorded force in nano-newtons and the x axis is the relative piezo displacement in nm.

FIG. 2.

A representative F-D curve (approach and retract) obtained in the experiment on onion epidermis cell wall in liquid. The y axis is the recorded force in nano-newtons and the x axis is the relative piezo displacement in nm.

Close modal

1. Contact mechanics models

Various contact mechanics can be used to describe the deformation of solids that touch each other. Here, we introduce briefly four types of frequently used contact mechanics models. Elastic deformation involving geometry effect was first studied with the Hertzian theory in 1881.44 This theory describes the elastic deformation properties of the materials through the circular contact area of a sphere with a plane.45 It assumes that there is no surface interaction such as Van der Waals force or adhesive forces. Other theories have been developed based on Hertz model. JKR (Johnson, Kendall, Roberts) theory considers the adhesive contacts.46 This model includes only a short-range adhesion in the contact area. Similar as the Hertzian theory, the JKR theory also assumes elastic sphere-sphere contacts. DMT theory later considers Van der Waals interactions outside the elastic contact regime. The JKR theory is applicable to large radius, compliant solids; while DMT theory applies to small radius, rigid solids.47,48 MD (Maugis-Dugdale) model considers intermediate states between JKR and DMT models.49 

DMT model works well for hard samples analyzed by AFM with a very sharp tip and the model is easy to use. For soft materials with high compliances, JKR model is more accurate, but data analysis is complicated. MD model works for all kinds of samples, but it is difficult to make an automated data processing algorithm. For these reasons, DMT model is usually used in the commercial software.

2. Built-in function of DMT modulus in peak force QNM mode

PeakForce QNM (Quantitative NanoMechanics) is an extension of PeakForce Tapping™ mode. The PeakForce Tapping operates similarly to a tapping mode with tip intermittently contacting the sample. However, PeakForce Tapping operates in a non-resonant mode. PeakForce Tapping mode modulates the Z-piezo at ∼2 kHz with default amplitude of 150 nm. PeakForce Tapping performs a very fast force curve analysis at every pixel in the image. A diagram of force curve for multiple mechanical properties analysis is shown in Figure 3. To obtain the Young's modulus, part of the retrace curve is fit using DMT model.50 DMT modulus map of a sample surface can be generated automatically during topography imaging.

FIG. 3.

Diagram of a force curve generated at 1 pixel during Peak Force QNM operation. Multiple mechanical properties such as modulus, adhesion, and deformation are derived from the force curve on the fly and mapped along with the topography images.

FIG. 3.

Diagram of a force curve generated at 1 pixel during Peak Force QNM operation. Multiple mechanical properties such as modulus, adhesion, and deformation are derived from the force curve on the fly and mapped along with the topography images.

Close modal

The reduced Young's modulus E* can be calculated according to the DMT model

FFadh=43E*Rd3.

FFadh is the force on the cantilever relative to the adhesion force. R is the tip radius and d is sample deformation.

The reduced modulus is related to the sample modulus by the following equation:

E*=[1νs2Es+1νtip2Etip]1,

where υs and Es are the Poisson's ratio and elastic modulus of the sample, respectively. υtip and Etip are the Poisson's ratio and elastic modulus of the tip, respectively.

As mentioned above, DMT model is applicable for hard samples. However, it may not be accurate for studying hydrated plant cell walls with very low modulus in aqueous solution. When the hydrophilic surface is analyzed in aqueous solution, adhesion force is negligible, as shown in Figure 2, so the simple Hertzian contact model should work better for analysis of nanoindentation data of hydrated cell walls.

The Hertz model assumes that the sample is isotropic, elastic and occupies infinite half space. It also assumes that the indenter is not deformable and there is no additional interaction between the tip and sample.51 Because the cell wall polymers are hydrophilic, the adhesion between cell walls and clean SiO2 tip surface in water is very small. Thus, a simple Hertz model can be used for F-D curve analysis to obtain the elastic modulus E.

As shown in Figure 4, when using the AFM tip to indent the sample, the recorded piezo displacement D from the free standing position is comprised of two parts: the tip cantilever bending x and the sample indentation δ.

D=x+δ.
(1a)

According to Hertz model, the force measured with a four-sided pyramid can be represented as52 

F=E1ν2tanα2δ2,
(1b)

where F is the recorded force, E is sample's elastic modulus, ν is sample's Poisson ratio, α is the geometry angle of the indenter. In our analysis, ν can be assumed to be 0.3 and α is estimated as 13° according to the parameters given by the manufacturer.53 

FIG. 4.

Sketch of indentation distance and tip geometry.

FIG. 4.

Sketch of indentation distance and tip geometry.

Close modal

Knowing that x=F/k, where k is the cantilever spring constant, from the above two equations, we can easily obtain relationship between recorded force F and recorded piezo displacement D

F=E1ν2tanα2(DFk)2.
(1c)

By fitting the experimental F-D retrace curve, we can get the elastic modulus E. Figure 5 shows a sample of F-D curve fitting result and it can be seen that the experimental data from AFM retrace curve and fitted data from the Hertz model are in good agreement.

FIG. 5.

A sample of F-D curve fitting. Experiment data in blue dots and fitted data in the red curve from the Hertzian model showed good agreement.

FIG. 5.

A sample of F-D curve fitting. Experiment data in blue dots and fitted data in the red curve from the Hertzian model showed good agreement.

Close modal

The never-dried cell wall thickness of the abaxial epidermal cells was determined with optical microscopy. The onions used in this study typically contained 12 scales. The outermost fresh scale will be referred as the 1st scale with the inner scales numbered sequentially through this paper. The epidermal cell wall thickness was measured from the 2nd, 5th, 8th, and 11th scales of three different bulbs. Figure 6 shows the cross-section images of the abaxial side of these hydrated onion scales. The thickness results are shown in Table I. The cell wall thickness increases from inner/younger scales to outer/older scales. The minimum thickness (∼2.4 μm), found in the 11th scale, is larger than ten times of the AFM indentation depth, a requirement for accurate indentation results.54 

FIG. 6.

Toluidine blue staining of hydrated onion scales cross section from (a) 2nd layer, (b) 5th layer, (c) 8th layer, and (d) 11th layer.

FIG. 6.

Toluidine blue staining of hydrated onion scales cross section from (a) 2nd layer, (b) 5th layer, (c) 8th layer, and (d) 11th layer.

Close modal
TABLE I.

Hydrated onion epidermis wall thickness measured by toluidine test.

Onion scalesCell wall thickness (μm)
AVG ± SEM (n = 3)
11.5 ± 0.7 
6.1 ± 0.9 
3.4 ± 0.7 
11 2.4 ± 0.7 
Onion scalesCell wall thickness (μm)
AVG ± SEM (n = 3)
11.5 ± 0.7 
6.1 ± 0.9 
3.4 ± 0.7 
11 2.4 ± 0.7 

Elastic moduli (E) of the abaxial epidermis walls from four scales (11th, 8th, 5th, and 2nd scales) in different onions were measured by AFM nanoindentation. At least 100 curves in 5 random cells in each scale were generated and analyzed. The results are shown in Figure 7. The moduli increase slightly from inner to outer scale for onions 1 and 2 but not in the other two onions. Unlike the cellulose microfibril alignment trend observed in the previous work, the indentation modulus shows no definitive trend from younger/inner to older/outer scales.34 

FIG. 7.

Elastic moduli of the 11th, 8th, 5th, and 2nd scales in four different onions. Standard deviations are shown with error bars. Changes in moduli are statistically significant.

FIG. 7.

Elastic moduli of the 11th, 8th, 5th, and 2nd scales in four different onions. Standard deviations are shown with error bars. Changes in moduli are statistically significant.

Close modal

Earlier it was hypothesized that the local density and orientation of cellulose microfibrils and/or pectin network status would be main factors contributing to the mechanical properties. It is difficult to alter artificially the microfibril density in the cell wall but is much easier to modify pectin networks. In this section, pectin networks were modified with chemical treatments and modulus was investigated to verify our assumption.

Pectin is a gel phase that embeds the cellulose-hemicelluloses network and becomes soluble with mild treatments. It serves as matrix material for the cell wall. Pectin accounts for a large proportion of onion cell wall in weight (about 42%).55 We test the effect of pectin network structure on modulus through control experiments. We chose two methods to modify the pectin network: ethylenediaminetetraacetic acid (EDTA) to partial removal of pectin and Ca2+ to cross-link pectin network.

As shown in Figure 7, the bulb-to-bulb variation was larger than the scale-to-scale variation for the same bulb. It is understandable that different bulbs would have different properties due to different growth environments and status. In our subsequent experiments, the “same” samples were measured before and after any treatments to avoid original modulus variation.

1. EDTA effect

In order to partially remove the EDTA-soluble pectin from the epidermis walls, 100 mM EDTA was added onto the cell wall sample taken from the 5th onion scale.56,57 Topography images were generated in the same area in water and after adding the EDTA solution. 80 F-D curves were probed in the sample area. Topography images and elastic moduli results are shown in Figure 8.

FIG. 8.

Topography images of onion 5th scale in (a) water, (b) 20 min after dipping in 100 mM EDTA, (c) 40 min after dipping in 100 mM EDTA, (d) 100 min after dipping in 100 mM EDTA. (e) Elastic moduli measured in water and after adding EDTA solution in water and after adding EDTA solution in the same area of cell wall shown in topography images. The error bar shows sample standard deviation.

FIG. 8.

Topography images of onion 5th scale in (a) water, (b) 20 min after dipping in 100 mM EDTA, (c) 40 min after dipping in 100 mM EDTA, (d) 100 min after dipping in 100 mM EDTA. (e) Elastic moduli measured in water and after adding EDTA solution in water and after adding EDTA solution in the same area of cell wall shown in topography images. The error bar shows sample standard deviation.

Close modal

Topography images became clearer with time after adding in 100 mM EDTA solution. Elastic modulus decreased slightly and remained stable with time after adding EDTA solution. The variation is statistically significant (in student's t-test, p = 0.04). Elastic modulus decrease with EDTA treatment is probably due to the removal of EDTA-soluble pectin.

2. Calcium ion effect

100 mM CaCl2 solution was added onto the cell wall removed from the 5th onion scale. Topography images were generated in the same area in water and after adding CaCl2 solution. 120 F-D curves were generated in the same area. Topography images and elastic moduli results are shown in Figure 9.

FIG. 9.

Topography images of 5th scale in (a) water, (b) 20 min after adding 100 mM CaCl2, (c) 30 min dipping in CaCl2 solution, (d) 40 min in CaCl2 solution, (e) 50 min in CaCl2 solution, and (f) 60 min in CaCl2 solution. (g) Elastic moduli of onion cell wall from 5th scale in water and in CaCl2 solution with time. The increase is statistically significant.

FIG. 9.

Topography images of 5th scale in (a) water, (b) 20 min after adding 100 mM CaCl2, (c) 30 min dipping in CaCl2 solution, (d) 40 min in CaCl2 solution, (e) 50 min in CaCl2 solution, and (f) 60 min in CaCl2 solution. (g) Elastic moduli of onion cell wall from 5th scale in water and in CaCl2 solution with time. The increase is statistically significant.

Close modal

Topography images showed apparent changes after adding 100 mM CaCl2 solution. Since pectin, especially homoglacturonan (HG) is negatively charged under cell wall physiological pH (around 5.0), Ca2+ serves as a ionic bridge that cross-links the HG chains, which adds rigidity to the cell wall.58,59 Due to the formation of gelation structures, elastic modulus increased dramatically after adding CaCl2 solution. The standard deviation increased significantly on the calcium ion treated sample. The topography and elastic modulus remained relatively stable with time.

Figure 10 shows the (a) nanoindentation results and (b) build-in DMT modulus mapping of calcium ion treated onion epidermis wall. The brightness of (a) reflects the height of the topography and that of (b) reflects the modulus. From the images, it can be seen that the DMT modulus mapping results are conformed to the nanoindentation results, although there are some differences in the actual values. For example, point 3 in topography image has large modulus (41.12 MPa) and corresponding point in DMT modulus mapping also has relatively high modulus (around 27 MPa) while point 7 in (a) has lower modulus (11.56 MPa) and it is also reflected in (b). This validates that the build-in DMT modulus mapping in Peak Force QNM mode provides reasonable qualitative reference for sample modulus analysis. The exact values, however, deviate from those determined with the Hertzian contact model, which is more appropriate for the hydrated cell walls in aqueous solutions. The most significant source of errors would be the uncertainty of the tip radius, which is typically taken from the nominal value provided by the AFM tip supplier, rather than determined from accurate measurements. Also any dynamic changes during the short contact/impact time between tip and sample would cause errors in the deformation depth at a given peak force operation condition.

FIG. 10.

(a) Topography image of calcium ion treated onion epidermis wall showing targeted nanoindentation results. (b) DMT modulus mapping of the same area. Image size is 2 × 2 μm.

FIG. 10.

(a) Topography image of calcium ion treated onion epidermis wall showing targeted nanoindentation results. (b) DMT modulus mapping of the same area. Image size is 2 × 2 μm.

Close modal

The mechanical properties of plant cell walls in their natural hydrated state are critical in understanding cell wall structure and cell growth. Indentation technique can provide valuable information in this regard. However, in indentation tests, the indentation depth needs to be less than 10% of the sample thickness so that the substrate effect becomes negligible. Our thickness measurements of onion epidermis cell wall indicated that the wall thickness in younger scales can be as thin as around 2 μm. This means accurate measurements would require the indentation depth to be 200 nm or less, making the conventional indentation technique unsuitable. We therefore conducted nanoindentation tests with AFM, allowing the indentation depth to be controlled below 150 nm. The AFM based nanoindentation also provided the ability to operate in liquid environment so that the cell walls can be studied in their natural, fully hydrated state.

The elastic modulus of single layered, fully hydrated abaxial epidermis walls from four scales (11th, 8th, 5th, and 2nd scales) in four onions were measured by AFM nanoindentation. The moduli increase slightly from inner to outer scale for two onions in experiments but not in the other two. From the view of composite mechanics, the indentation was measured along the direction normal to the microfibril layers and caused very small lateral displacement. Thus, the cellulose microfibril orientation would influence more the in-plane tensile stress/strain anisotropy than the out-of-plane modulus. Although the net orientation of cellulose microfibrils varies gradually from the dispersed arrangement in the inner scales to the transverse orientation in the outer scales as shown in our previous study,34 the nanoindentation moduli of cell walls do not show substantial change that can be correlated with such variation. This indicates that, for the onion epidermis cell walls, the cellulose microfibril orientation alone may not be the dominating factor determining the out of plane modulus. Other factors such as the local density of microfibrils and pectin network status could make more significant contribution to the nanoindentation modulus.

Pectin forms a gel phase structure that holds cellulose-hemicelluloses frame in cell walls. From the mechanical point of view, pectin serves as a matrix material in the cell wall biopolymer composite and has long been suspected of playing an important role in cell wall mechanics. Since our AFM based nanoindentation was operated in liquid, the sample chemical environment can be controlled and modified. This allowed us to investigate the effect of pectin network structure on wall mechanical property. Two methods were used to modify the pectin network: EDTA to partial remove and Ca2+ to cross-link the pectin network.

These two methods caused different effects on the cell wall property. In particular, Ca2+ treatment caused the formation of cross-linked pectin gelation structures, which can be clearly observed in the topography images in Figure 9. The elastic moduli were found to increase dramatically as a result. On the other hand, the topography images after the EDTA treatment became clearer as a result of surface pectin removal. Correspondingly, the moduli decreased, indicating the weakening of pectin structure. This verifies that the pectin network has significant impact on cell wall property. In plant, the pectin network could be altered by various factors such as the environment, age, etc., which would substantially affect the cell wall mechanical property. This is an interesting topic that warrants future studies.

Moduli measured by nanoindentation in epidermis cell walls of onion scales indicate that microfibril orientation may not be a governing contribution to cell wall modulus. Control experiments on pectin network on the other hand showed that the pectin can substantially impact the indentation modulus. The AFM based nanoindentation can effectively track variations in cell wall properties, demonstrating the feasibility of this technique as a tool to characterize intact plant cell walls in their fully hydrated state.

This work was supported as part of the Center for Lignocellulose Structure and Formation (CLSF) an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, and Office of Basic Energy Sciences under Award No. DE-SC0001090. The authors acknowledge Kabindra Kafle for his help with the cell wall thickness measurements as well as Tian Zhang, Liza Hall, Yong Bum Park, and Ed Wagner for technical support.

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