Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

Summary

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

Abstract

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.

Introduction

Atomic force microscopy (AFM) belongs to the scanning probe microscopy (SPM) family, where a tip with a radius of usually a few nanometers scans the surface of a sample. The detection of a surface is not achieved via optical or electron-based methods, but via the interaction forces between the tip and the sample surface. Thus, this technique is not limited to topographic characterization of a sample surface (3D resolution which can go down to a few nanometers), but also allows the measurement of any type of interaction forces such as electrostatic, van der Waals or contact forces. Furthermore, the tip can be used to apply forces at the surface of a biological sample a....

Protocol

1.Measure of Cell Wall Mechanical Properties

NOTE: Example of the developing gynoecium of Arabidopsis is presented.

1. Preparation of the biological samples
   1. Collect a closed flower bud at stage 9 to 10 (about 0.5 mm long) according to published stages determination for Arabidopsis⁴. Under a binocular, using fine tweezers, carefully open the bud to check the stage of development and collect the gynoecium located at the center of the flower.
   2. Put the gynoecium on double side tape placed at the center of the cover of a small Petri dish (diameter o....

Results

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 surfa.......

Discussion

The emergence of shapes in plants is mainly determined by the coordinated rate and direction of growth during time and space. Plant cells are encased in a rigid cell wall made of a polysaccharidic matrix, which glues them together. As a result, cell expansion is controlled by the equilibrium between turgor pressure pulling on the cell wall, and stiffness of the cell wall resisting to this pressure. In order to understand the mechanisms underlying development, it is important to be able to measure both cell wall mechanica.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Growth medium
1,000x 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, 2 mM.
MURASHIGE & SKOOG MEDIUM	Duchefa Biochimie	M0221.0025	Basal salt mixture, used to make ACM, 2.2 g/ 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.