Name: use of atomic force microscopy to measure mechanical properties and turgor pressure of plant cells and plant tissues
Uploaded: 2019-07-15T15:00:00
Description: Watch this Scientific Journal Video about Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues at JoVE.com

We would like to thank the PLATIM team for their technical support, as well as Arezki Boudaoud and members of the Biophysic team at the RDP lab for helpful discussions.

1.Measure of Cell Wall Mechanical Properties
NOTE: Example of the developing gynoecium of Arabidopsis is presented.
<ol>
	<li>Preparation of the biological samples
	<ol>
		<li>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 loc...

<ol>
	<li>Du, F., Guan, C., Jiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+leaf+morphogenesis.">Molecular mechanisms of leaf morphogenesis.</a> Molecular Plant. 11, 1117-1134 (2018).</li><li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+the+plant+cell+wall.">Growth of the plant cell wall.</a> Nature Reviews Molecular Cell Biology. 6, 850-861 (2005).</li><li>Dumais, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+mechanics+control+pattern+formation+in+plants?.">Can mechanics control pattern formation in plants?.</a> Current Opinion in Plant Biology. 10, 58-62 (2007).</li><li>Smyth, D. R., Bowman, J. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+flower+development+in+Arabidopsis.">Early flower development in Arabidopsis.</a> The Plant Cell. 2, 755-767 (1990).</li><li>Routier-Kierzkowska, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+force+microscopy+for+in+vivo+measurements+of+plant+tissue+mechanics.">Cellular force microscopy for in vivo measurements of plant tissue mechanics.</a> Plant Physiology. 158 (4), 1514-1522 (2012).</li><li>Corson, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+a+plant+tissue+into+a+living+cell+froth+through+isotropic+growth.">Turning a plant tissue into a living cell froth through isotropic growth.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 8453-8458 (2009).</li><li>Hervieux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanical+feedback+restricts+sepal+growth+and+shape+in+Arabidopsis.">A mechanical feedback restricts sepal growth and shape in Arabidopsis.</a> Current Biology. 26, 1019-1028 (2016).</li><li>Stanislas, T., Hamant, O., Traas, J., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11+-+In-vivo+analysis+of+morphogenesis+in+plants.">Chapter 11 - In-vivo analysis of morphogenesis in plants.</a> Methods in Cell. 139, 203-223 (2017).</li><li>Beauzamy, L., Derr, J., Boudaoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+hydrostatic+pressure+in+plant+cells+using+indentation+with+an+atomic+force+microscope.">Quantifying hydrostatic pressure in plant cells using indentation with an atomic force microscope.</a> Biophysical Journal. 108 (10), 2448-2456 (2015).</li><li>Costa, K. D., Sim, A. J., Yin, F. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Hertzian+Approach+to+Analyzing+Mechanical+Properties+of+Endothelial+Cells+Probed+by+Atomic+Force+Microscopy.">Non-Hertzian Approach to Analyzing Mechanical Properties of Endothelial Cells Probed by Atomic Force Microscopy.</a> Journal of Biomechanical Engineering. 128 (2), 176-184 (2006).</li><li>Beauzamy, L., Louveaux, M., Hamant, O., Boudaoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically,+the+shoot+apical+meristem+of+Arabidopsis+behaves+like+a+shell+inflated+by+a+pressure+of+about+1MPa.">Mechanically, the shoot apical meristem of Arabidopsis behaves like a shell inflated by a pressure of about 1MPa.</a> Frontiers in Plant science. 6 (1038), 1-10 (2015).</li><li>Majda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanochemical+polarization+of+contiguous+cell+walls+shapes+plant+pavement+cells.">Mechanochemical polarization of contiguous cell walls shapes plant pavement cells.</a> Developmental Cell. 43 (3), 290-304 (2017).</li><li>Torode, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branched+pectic+galactan+in+phloem-sieve-element+cell+walls:+implications+for+cell+mechanics.">Branched pectic galactan in phloem-sieve-element cell walls: implications for cell mechanics.</a> Plant Physiology. 176, 1547-1558 (2018).</li><li>Farahi, R. 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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...

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 and measure the resulting deformation, the so-called &#34;indentation&#34;, in order to determine its mechanical properties (e.g., Young&#39;s modulus, viscoelastic properties).
Mechanical properties of plant cell walls are essential to be taken into account when trying to understand mechanisms underlying developmental processes1,2,3. Indeed, these properties are tightly controlled during development, in particular since cell wall softening is required to allow cells to grow. AFM can be used to measure these properties and study the way they change between organs, tissues or developmental stages.
In this paper, we describe how we use AFM to measure both cell wall mechanical properties and turgor pressure. These two applications are demonstrated on two different AFM microscopes and are detailed here after.

<table border="0" cellpadding="0" cellspacing="0" style="width:700px;" width="700">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Growth medium</td>
			<td style="width:155px;"></td>
			<td style="width:151px;"></td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">1000x vimatin stock solution</td>
			<td style="width:155px;"></td>
			<td style="width:151px;"></td>
			<td>used to make ACM, composition see Stanislas et al., 2017. Add to ACM after autoclaving, before pouring.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">1-N-Naphthylphthalamic acid (NPA)</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">132-66-1</td>
			<td>add to Arabidopsis medium, 10 &#956;M. Add after autoclaving, before pouring.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">agar-agar</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">9002-18-0</td>
			<td>add to Arabidopsis medium, 1% w/v.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">agarose</td>
			<td style="width:155px;">Merck Millipore</td>
			<td style="width:151px;">9012-36-6</td>
			<td>used to make solid ACM, 0.8% w/v.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Arabidopsis medium</td>
			<td style="width:155px;">Duchefa Biochimie</td>
			<td style="width:151px;">DU0742.0025</td>
			<td>For in vitro arabidopsis culture, 11.82g/L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Calcium nitrate tetrahydrate</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">13477-34-4</td>
			<td>add to Arabidopsis medium, 2mM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">MURASHIGE &#38; SKOOG MEDIUM</td>
			<td style="width:155px;">Duchefa Biochimie</td>
			<td style="width:151px;">M0221.0025</td>
			<td>Basal salt mixture, used to make ACM, 2.2g/L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">N6-benzyladenine (BAP)</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">1214-39-7</td>
			<td>used to make ACM, 555 nM. Add to ACM after autoclaving, before pouring.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">oryzalin</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">19044-88-3</td>
			<td>for oryzalin treatement, 10 &#956;g/mL.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">plant preservation mixture (PPM)</td>
			<td style="width:155px;">Plant Cell Technology</td>
			<td style="width:151px;"></td>
			<td>used to make ACM, 0.1% v/v. Add to ACM after autoclaving, before pouring.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Potassium hydroxide</td>
			<td style="width:155px;">Duchefa Biochimie</td>
			<td style="width:151px;">1310-58-3</td>
			<td>used to make Arabidopsis medium and ACM, both pH 5.8.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">sucrose</td>
			<td style="width:155px;">Duchefa Biochimie</td>
			<td style="width:151px;">57-50-1</td>
			<td>used to make ACM, 1% w/v.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tools for AFM</td>
			<td style="width:155px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">BioScope Catalyst BioAFM</td>
			<td style="width:155px;">Bruker</td>
			<td style="width:151px;"></td>
			<td>The AFM used for turgor pressure measurement in this protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Nanowizard III + CellHesion</td>
			<td style="width:155px;">JPK (Bruker)</td>
			<td style="width:151px;"></td>
			<td>The AFM used for measuring mechanical properties.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Patafix</td>
			<td style="width:155px;">UHU</td>
			<td style="width:151px;">D1620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Reference elasitic structure</td>
			<td style="width:155px;">NanoIdea</td>
			<td style="width:151px;">2Z00026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Reprorubber-Thin Pour</td>
			<td style="width:155px;">Flexbar</td>
			<td style="width:151px;">16135</td>
			<td>biocompatible glue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Spherical AFM tips</td>
			<td style="width:155px;">Nanoandmore</td>
			<td style="width:151px;">SD-SPHERE-NCH-S-10</td>
			<td>Tips used for measuring mechanical properties.</td>
		</tr>
	</tbody>
</table>

use of atomic force microscopy to measure mechanical properties and turgor pressure of plant cells and plant tissues

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.

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...

Watch this Scientific Journal Video about Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues at JoVE.com

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

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.

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

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