AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

Summary

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Abstract

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK AFM. Specifically, in this protocol we measure the apparent Young’s modulus of cell walls at subcellular resolutions across regions of up to 100 µm x 100 µm in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.

In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.

However, several limitations remain: the method can only be used on fairly small samples (around 100 µm in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue’s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

Introduction

Growth in plants is achieved by the coordinated expansion of the rigid cell walls that surround each and every cell of the organism. Accumulating evidence indicates that it is through the modification of cell wall chemistry that plants locally control this expansion. The expansion is thought to be driven primarily by strain on the cell walls, caused by the cell’s high turgor pressure; this strain response to turgor pressure is governed by the mechanical properties of the cell walls¹. Little is known of these mechanical properties and how they change during development. Furthermore little is known of how these mechanical properties are controlled and w....

Protocol

1. Prepare Glass Slides for Mounting Sample

1. Prepare imbedding agarose media: 0.7% low melting agarose in 10% mannitol (in water).
2. Using a strong metal instrument (e.g. drill tip, lime), etch out a 0.5 x 0.5 cm area in the center of a microscopy glass slide. Or instead, glue a small piece of glass lamella (about 20 x 200 µm) to the glass slide using Araldite glue. NOTE: This roughens the surface in order to facilitate the adhesion of the agarose to ensure that the agarose media sticks or fixes to the slide. This slide can be reused.
3. Position a droplet of about 20 µl of imbedding agarose media o....

Results

In Figure 1 we present typical Young moduli maps of floral meristems (Figures 1A and 1B), young and old hypocotyls (Figures 1C-F), and root meristem (Figure 1G and 1H). In all experiments the indenter is hemispherical, but its radius differs so that different spatial resolutions can be achieved. Figures 1C and 1D show typical results for meso-nanoscale indenters (50 nm radius) with meso-.......

Discussion

In plants, changing mechanical properties play a major role in directing growth and morphogenesis. To date there has been great progress in unraveling the genetic and chemical networks that control plant growth, but our knowledge of how these networks contribute to and are affected by changes in mechanical properties is rudimentary. This method should enable us to fill this gap, and so it should be of strong interest to scientists studying any aspect of plant growth or morphogenesis. We now summarize the challenges and l.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AFM	JPK	NanoWizard	All the 3-generation are able to do the work with the same preferment.
AFM stage	JPK	CellHesion	Required for sample with low topography (less than 11 µm between the lowest and the highest point in the area of force scanning).
AFM optics	JPK	Top View Optics	Very important in order to position the sample. Could be replaced by long range binoculars or a microscope.
Stereo microscope	Leica	M125	Any type of stereo microscope could do.
150 nm mounted cantilever	Nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel, Switzerland	R150-NCL-10	To measure only the cell wall at the surface of the epidermis use.
1 µm mounted cantilever	Nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel, Switzerland	SD-Sphere-NCH-S-10	To measure the mechanics of the cell wall orthogonal to the surface of the epidermis.
Tipless cantilever	Nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel, Switzerland	TL-NCH-20	To measure the local mechanics of the tissue (2-3 cell wide) use a 5 µm mounted cantilever. We attached a 5 µm borosilicate bead to a tipless cantilever.
5 µm silicon microspheres	Corpuscular	C-SIO-5
Araldite	Bartik S.A. 77170 Coubet, France		Araldite for fixing the bead to the tipless cantilever.
Low melting agarose	Fisher Scientific Fair Lawn, New Jersey 07410	BP160-100	34-45 °C gelation temperature
D-Mannitol	Sigma-Aldrich, 3050 Spruce Street, St. Louis, MO 63103 USA	M4125-500G
2 Stainless Steel No. 5 Tweezers	Ideal-Tek 6828 Balerna, Switzerland	951199