We would like to acknowledge Dr. Dmitry Suslov (Saint Petersburg State University, Saint Petersburg, Russia) and Prof. Mira Ponomareva (Tatar Scientific Research Institute of Agriculture, FRC KazSC RAS, Kazan, Russia) for providing maize and rye seeds, respectively. The presented method was developed within the framework of the Russian Science Foundation Project No. 18-14-00168 awarded to LK. The part of the work (obtaining of the results presented) was performed by AP with the financial support of the government assignment for the FRC Kazan Scientific Center of RAS.

1. Sample preparation for AFM measurements
<ol>
	<li>Plant material: Sterilize the seeds of maize (Zea mays L.) and rye (Secale cereale L.) with a 0.35% NaOCl solution for 10 min, wash 3x with distilled water, and then grow hydroponically in the dark at 27 &#176;C for 4 days and 2 days, respectively. Primary roots were used for the experiment.</li>
	<li>Preparation of solutions and sample for vibratome sectioning
	<ol>
		<li>Prepare agarose solution for root embedding by dissolving 3%...

<ol>
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E., 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=Mapping+nano-scale+mechanical+heterogeneity+of+primary+plant+cell+walls.">Mapping nano-scale mechanical heterogeneity of primary plant cell walls.</a> Journal of Experimental Botany. 67 (9), 2799-2816 (2016).</li><li>Zdunek, A., Kurenda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+elastic+properties+of+tomato+fruit+cells+with+an+atomic+force+microscope.">Determination of the elastic properties of tomato fruit cells with an atomic force microscope.</a> Sensors. 13 (9), 12175-12191 (2013).</li><li>Evered, C., Majevadia, B., Thompson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+water+content+has+a+direct+effect+on+extensibility+in+growing+hypocotyls+of+sunflower+(Helianthus+annuus+L).">Cell wall water content has a direct effect on extensibility in growing hypocotyls of sunflower (Helianthus annuus L).</a> Journal of Experimental Botany. 58 (12), 3361-3371 (2007).</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 (2), 1547-1558 (2018).</li><li>Garcia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+mapping+of+soft+materials+with+the+atomic+force+microscope:+methods,+theory+and+applications.">Nanomechanical mapping of soft materials with the atomic force microscope: methods, theory and applications.</a> Chemical Society Reviews. 49 (16), 5850-5884 (2020).</li><li>Kozlova, L., Petrova, A., Ananchenko, B., Gorshkova, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+primary+cell+wall+nanomechanical+properties+in+internal+cells+of+non-fixed+maize+roots.">Assessment of primary cell wall nanomechanical properties in internal cells of non-fixed maize roots.</a> Plants-Basel. 8 (6), 172 (2019).</li><li>Bovio, S., Long, Y. C., Moneger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+atomic+force+microscopy+to+measure+mechanical+properties+and+turgor+pressure+of+plant+cells+and+plant+tissues.">Use of atomic force microscopy to measure mechanical properties and turgor pressure of plant cells and plant tissues.</a> Journal of Visualized Experiments. (149), e59674 (2019).</li><li>Krieg, 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=Atomic+force+microscopy-based+mechanobiology.">Atomic force microscopy-based mechanobiology.</a> Nature Reviews Physics. 1 (1), 41-57 (2019).</li><li>Braunsmann, C., Schaffer, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Note:+Artificial+neural+networks+for+the+automated+analysis+of+force+map+data+in+atomic+force+microscopy.">Note: Artificial neural networks for the automated analysis of force map data in atomic force microscopy.</a> Review of Scientific Instruments. 85 (5), 056104 (2014).</li></ol>

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth, and therefore the future size and shape of the plant. The AFM-based method presented here complements existing techniques which are used to study the properties of plant cell walls. It allows the elasticity of cell walls, which belong to the inner tissues of the plant, to be investigated. Using the presented method, the mechanical properties of cell walls in different tissues of the growing maize root were mapped, a...

The mechanical properties of the plant cell wall determine the shape of the cell and its ability to grow. For example, the growing tip of the pollen tube is softer than the non-growing parts of the same tube1. The primordia formation on Arabidopsis meristem is preceded by a local decrease in cell wall stiffness at the site of the future primordium2,3. The cell walls of Arabidopsis hypocotyl, which are parallel to the main growth axis and grow faster, are softer than those that are perpendicular to this axis and grow slower4,5. In the maize root, the transition of cells from division to elongation was accompanied by a decrease in elastic moduli in all tissues of the root. The moduli remained low in the elongation zone and increased in the late elongation zone6.
Despite the availability of various methods, the large arrays of biochemical and genetic information on cell wall biology obtained annually are rarely compared with the mechanical properties of cell walls. For example, mutants on cell wall-related genes often have altered growth and development4,7,8, but are rarely described in terms of biomechanics. One of the reasons for this is the difficulty of conducting measurements at the cellular and subcellular levels. Atomic force microscopy (AFM) is currently the primary approach for such analyses9.
In recent years, numerous AFM-based studies on plant cell wall biomechanics have been carried out. The mechanical properties of cell walls of the outer tissues of Arabidopsis2,3,4,5,10,11 and onion12, as well as of cultured cells13,14,15, have been investigated. However, the superficial cells of a plant may have cell walls whose mechanical properties differ from those of the inner tissues6. In addition, plant cells are pressurized by turgor which makes them stiffer. To get rid of the influence of turgor pressure, researchers have to use plasmolyzing solutions2,3,4,5,10,11 or decompose the values obtained into turgor and cell wall contributions12. The first approach leads to sample dehydration and changes the thickness and properties of the cell wall16, while the second approach requires additional measurements and complicated mathematics, and applies only to cells of relatively simple shape12. The cell wall properties of internal tissues can be evaluated on cryosections17 or sections of plant material impregnated with resin8. However, both methods involve dehydration and/or impregnation of samples, which inevitably leads to changes in properties. The properties of isolated or cultured cells are difficult to relate to the physiology of the whole plant. Both cultivation and isolation of plant cells can affect the mechanical properties of their cell walls.
The method presented here complements the aforementioned approaches. Using it, the primary cell walls of any tissue and at any stage of plant development can be examined. Sectioning and AFM observations were performed in liquid which avoids sample dehydration. The problem of turgor was solved as the cells are cut. The protocol describes work with maize and rye roots, but any other sample can be examined if it is suitable for vibratome sectioning.
The AFM studies described here were performed using the force-volume technique. Different instruments use different names for this method. However, the basic principle is the same; a force-volume map of the sample is obtained by a sinusoidal or triangular motion of the cantilever (or sample) to achieve a certain loading force at each analyzed point, while recording the cantilever deflection18. The result combines a topographic image of the surface and the array of force-distance curves. Each curve is used to calculate the deformation, stiffness, Young&#39;s modulus, adhesion, and energy dissipation at a specific point. Similar data can be obtained by point-by-point force-spectroscopy after scanning in contact mode19, although it is more time-consuming.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose, low melting point</td>
			<td>Helicon</td>
			<td>B-5000-0.1</td>
			<td>for sample fixation</td>
		</tr>
		<tr>
			<td>Brush</td>
			<td>-</td>
			<td>-</td>
			<td>for section moving</td>
		</tr>
		<tr>
			<td>Cantilevers</td>
			<td>NanoTools, Germany</td>
			<td>NT_B150_v0020-5</td>
			<td>Model: Biosphere B150-FM</td>
		</tr>
		<tr>
			<td>Cantilevers</td>
			<td>NT-MDT, Russia</td>
			<td>FMG01/50</td>
			<td>Model: FMG01</td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>-</td>
			<td>-</td>
			<td>for vibratomy</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Heinz Herenz</td>
			<td>1042000</td>
			<td>for vibratomy and AFM calibration</td>
		</tr>
		<tr>
			<td>ImageAnalysis P9 Software</td>
			<td>NT-MDT, Russia</td>
			<td>-</td>
			<td>for data analysis</td>
		</tr>
		<tr>
			<td>Leica DM1000 epifluorescence microscope</td>
			<td>Leica Biosystems, Germany</td>
			<td>11591301</td>
			<td>for section check</td>
		</tr>
		<tr>
			<td>NaOCl</td>
			<td>-</td>
			<td>-</td>
			<td>for seed sterilization</td>
		</tr>
		<tr>
			<td>Nova PX 3.4.1 Software</td>
			<td>NT-MDT, Russia</td>
			<td>-</td>
			<td>for experiments conducting</td>
		</tr>
		<tr>
			<td>NTEGRA Prima microscope with HD controller</td>
			<td>NT-MDT, Russia</td>
			<td>-</td>
			<td>for AFM and data acquisition</td>
		</tr>
		<tr>
			<td>Petri dish 35 mm</td>
			<td>Thermo Fisher Scientific</td>
			<td>153066</td>
			<td>for sample fixation</td>
		</tr>
		<tr>
			<td>Tip pipette 1000 &#181;L</td>
			<td>Thermo Fisher Scientific</td>
			<td>4642092</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tip pipette 2-20 &#181;L</td>
			<td>Thermo Fisher Scientific</td>
			<td>4642062</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vibratome Leica VT 1000S</td>
			<td>Leica Biosystems, Germany</td>
			<td>1404723512</td>
			<td>for sample sectioning</td>
		</tr>
	</tbody>
</table>

characterizing mechanical properties of primary cell wall in living plant organs using atomic force microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Typical elastic modulus and DFL maps, as well as force curves obtained on rye and maize roots by the method described, are presented in Figure&#160;2. Figure&#160;2A shows elastic modulus and DFL maps obtained on the transverse section of rye primary root. The white areas in the modulus map (Figure&#160;2A, left) correspond to an erroneous overestimation of Young&#39;s modulus due to the scanner reaching its limit in the z-direction...

Watch this Scientific Journal Video about Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy at JoVE.com

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

Plant cell growth is controlled by the mechanical properties of their cell walls. Therefore, it's important to know these properties in different organ and tissues of the plants. The method presented here allows the biomechanical characterization of non-fixed and non-dehydrated cell walls in the internal tissues of young plants.Start with preparing the solutions and sample for vibratome sectioning. Pour a four millimeter layer of melted 3%agarose on the bottom of the Petri dish and let it cool slightly to prevent thermal damage to the sample. Place about five millimeter long three or four pieces of the plant organ horizontally on the agarose.Around 30 to 60 seconds later, a thin semi-solid film will appear on top of the first agarose layer. Then, carefully pour a second layer on top. After the agarose is completely solidified, cut out the block containing the specimen.Shape the block into a hexagonal truncated pyramid to ensure its stability during further sectioning. Before starting the sample sectioning with the vibratome, glue the block to the vibratome stage with cyanoacrylate adhesive. Place the stage in the vibratome, so that one of the pyramid corners faces the blade of the vibratome and pour water into the vibratome bath.Set the sectioning parameters like section thickness, blade speed, and vibration frequency and cut the sample. Using a fine brush, move the section from the water bath onto a glass slide and place a drop of water on the section to prevent it from drying out. After checking the quality of the section under a light microscope, select the proper section having cell wall perpendicular to the section plane.Then immobilize the section for atomic force microscopy measurements by pouring a layer of one milliliter of 1%molten agarose on the bottom of the Petri dish cap using a pipette. After the agarose has solidified, remove excess water from the section by bringing filter paper to its edge. Carefully transfer the section from the slide to the center of the Petri dish cap using a brush.Then carefully add 1%agarose around the section using a 20 microliter pipette and pour the water or other solution for the atomic force microscopy into the Petri dish cap with the immobilized section. Guide the sample under the atomic force microscopy cantilever using the optical microscope. Click on the Approach button and then the Landing button to approach the sample in contact mode with a set point of one nanoampere.Click on the Scanning button and then the Area button. Select the area size of 50 micrometers by 50 micrometers to scan. Click on the Move Probe button and check the entire scan area by moving the scanner over it and finding the highest point based on the degree of scanner protrusion.Open the Approach tab. Then click on the Remove button to retract from the sample. Using the highest point as a target, click the Landing button to approach the sample again.Then check the surface again by clicking on the Move Probe button and moving the scanner over it. Set the scan rate to 0.5 hertz, and set the scan size to 50 micrometers by 50 micrometers and the scan point to 64 by 64. Click on the Run button and scan to check the surface of the sample and it's possible contamination with agarose.After clicking the On button, choose the HDPlus mode in the dropdown menu in the program's main window and set the set point to 0.1 nanoamperes in the main program window. In the main tab of the HD window, set the scanning parameters appropriate to the studied sample. Then open the Noises tab in the HD window and enter the cantilever's resonance frequency.Open the Quant tab of the HD window and enter the IOS, cantilever stiffness, tip radius, and angle. Select the model of contact, which will be used for calculations depending on tip geometry. After this, open the Scan tab of the HD window and select the signals, as well as the direction in which the signal is recorded.Tick the Force Volume box to get a record of all force curves and click on the Off button at the top of the main program window to switch on the feedback loop Click on the Phase Core button in the main HD window to correct the sensitivity of the optical system. The Versus Time tab of the main HD window presents the function of the DFL signal versus time in real time. Select the parts of this function that will be used for baseline level determination and to fit the contact model for further calculations.Now, set the scan point value to 256 by 256 in the main window of the program. Then, set the scan rate to 0.2 hertz and click on the Run button to scan the sample. After scanning stops, click on the On button at the top of the main window to switch off the feedback loop.Choose Contact Mode in the dropdown menu, open the Approach tab, and click on the Remove button to retract from the sample. Click on the Data button to open the analysis software and save the output. Open the saved file in the analysis software.Select the HD force volume frame. Hold down the Control key and select one visual frame obtained in the same scanning direction. Click on the Load External Map button to see where the cell walls are located.Check InvOptSens and cantilever stiffness values in the main tab. Open the Additional tab and check the tip parameters and contact model. Click on various points on the cell walls on the visual frame and select only those curves well-described by the model.The white areas in the modulus map correspond to an erroneous overestimation of Young's modulus due to the scanner reaching its limit in the Z direction. This image is not convenient to be used as an external map for a further selection of satisfactory force curves. However, the DFL signal map presented on the right is better suited here.Different instruments may refer to DFL signals as deflection or error signals. The scanner that has been fully extended while trying to reach the bottom of the sample can result in erroneous measurements and even interrupted scans. The agarose presence can be checked while obtaining the first scan in the contact mode as the cell walls and some cell bottoms are visible on such scans.However, in case of inaccurate immobilization, the surface may be covered with agarose, which masks the sample topography. Out of four different force curves recorded at different points of the same cell wall, the curve recorded at 0.1 shows no baseline, meaning no separation of the cantilever tip from the cell wall. The curve recorded at 0.3 shows a shoulder on the approaching part indicating bending of the cell wall.Points two and four show satisfactory force curves with similar moduli values. Sample preparation and examination require practice, but it can be mastered. A crucial part of this protocol is to filter resulting curves.Do not rely on moduli, which were calculated automatically. The same technique can be used to measure the adhesion forces or energy dissipation. It also can be important for description of viscose or viscoelastic behavior of some materials.

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1.9K 次观看.  Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS. 植物细胞的生长由其细胞壁的机械性能控制。因此，了解植物不同器官和组织中的这些特性非常重要。这里介绍的方法允许对幼苗内部组织中的非固定和非脱水细胞壁进行生物力学表征。首先准备用于振动切片的溶液和样品。在培养皿底部倒入四毫米的融化的3%琼脂糖层，使其稍微冷却以防止对样品的热损伤。将大约五毫米长的三到四块植物器官水平放在琼脂糖上。