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Imaging and Measurement of Nanomechanical Properties within Primary Xylem Cell Walls of Broadleaves
阔叶初生木质部细胞壁内的纳米力学性能的成像和测定   

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Abstract

A technique of atomic force microscopy (AFM) called PeakForce quantitative nanomechanical mapping (PeakForce QNM) is an efficient tool for the quantitative mechanobiological imaging of fibrillar aggregate, human epidermal cell and woody plant cell wall topography (Sweers et al., 2011; Heu et al., 2012; Ďurkovič et al., 2012; Ďurkovič et al., 2013). Here, we describe a detailed protocol for the measurement of nanomechanical properties of primary xylem cell walls in woody plants, for the determination of reduced Young’s modulus of elasticity (MOE), adhesion, deformation, and energy dissipation (Figure 1). This new technique provides direct control of the maximum loading force and the deformation depth in cell wall samples keeping indentations small, while at the same time eliminating damaging lateral forces in order to preserve both the AFM tip and plant sample. High-resolution and non-destructive imaging shed new quantitative mechanistic insights into the structural biology of woody plant cell walls. This procedure can also be adapted for other biological samples with a varying range of stiffness.

Keywords: Atomic force microscopy(原子力显微镜), Cell wall nanomechanics(细胞壁力学), Modulus of elasticity(弹性模量), Adhesion(粘附), PeakForce QNM(PeakForce QNM)


Figure 1. Basic principles illustrating how the different nanomechanical properties are extracted from the force curve. The image shows a typical force curve for a primary xylem cell wall sample in the Dutch elm disease-sensitive hybrid 'Groeneveld'. PeakForce QNM mode performs a very fast force curve at every pixel in the image. Analysis of force curve data is done on the fly, providing a map of multiple nanomechanical properties that has the same resolution as the height image. The adhesion is the vertical distance between the base line and the lowest portion of the retraction curve. The deformation is the horizontal distance between the contact point and the turn-away point (representing maximum indentation). The Young’s modulus of elasticity can be extracted by extrapolating the linear portion of the retraction curve after the contact point using a Derjaguin, Muller, Toporov (DMT) model fit. The slope of the linear portion is determined according to the least square method procedure. The energy dissipation can be calculated by integrating the area between the two curves.

Materials and Reagents

  1. Leaf midribs from broadleaves (Ulmus spp., Sorbus spp.)
  2. Glutaraldehyde solution, 25% in H2O (Grade II) (Sigma-Aldrich, catalog number: G6257 )
  3. Ethanol solutions (Analytical Reagents)
    Note: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, each of them prepared by diluting of 96% ethanol in a 0.1 M cacodylate buffer, and 100% ethanol.
  4. 25% glutaraldehyde (Grade I) (Sigma-Aldrich, catalog number: G5882 )
  5. Xylene (Analytical Reagents) or alternative cleaners with a lower toxicity (for example D-limonene)
    Note: Work with xylene or D-limonene only in a properly operating and certified fume cupboard.
  6. Paraplast plus (Sigma-Aldrich, catalog number: P3683 )
  7. (3-Aminopropyl) triethoxy-silane (Sigma-Aldrich, catalog number: A3648 )
  8. 0.1 M cacodylate buffer (see Recipes)
  9. Circle glass slides coated with (3-aminopropyl)triethoxy-silane (see Recipes)
  10. 1x phosphate buffered saline (PBS) (see Recipes)
  11. Glutaraldehyde fixative solution (see Recipes)

Equipment

  1. Razor blades (fine tweezers and heated forceps)
  2. pH meter
  3. Exicator with a vacuum pump
  4. Petri dishes
  5. Laboratory drying chamber
  6. Retracting base sledge microtome (Series 8000) (Bright Instrument Co.)
  7. Circle glass slides (11 mm in diameter, 1 mm thick)
  8. Slide warmer
  9. Steel AFM sample mounting disks
  10. Sample adhesive pads
  11. Silicon probes MPP-12120 (model: TAP150A ) having a 180° rotated tip and the spring constant at least 5 N/m (Bruker AFM Probes)
  12. Fused silica reference sample (nominal MOE 72.9 GPa) or any other reference sample having the MOE value higher than 70 GPa
  13. TGT1 commercial tip characterization grating (NT-MDT Co.)
  14. MultiMode 8 atomic force microscope with a Nanoscope V controller and a PeakForce Quantitative Nanomechanical Mapping mode (Bruker Corporation)

Software

  1. NanoScope analysis (version 1.40r2) (Bruker Corporation)
  2. MATLAB (version 7) (MathWorks)

Procedure

  1. Separate leaf midrib samples (0.3-0.4 cm x 0.3-0.4 cm) from the donor plants (Figures 2A-B).
  2. Immerse the samples into a fixative solution of 5% (v/v) glutaraldehyde (Grade I) in a 0.1 M cacodylate buffer at pH 7.2, and then place them in a vacuum for 15 min at room temperature to remove air from the leaf tissue. Fix the samples in a fixative solution for six hours at 4 °C.
  3. Dehydrate the samples in an ascending series of ethanol solutions for 20 min each step at room temperature, follow with mixtures of ethanol and xylene at the ratios 3:1, 1:1 and 1:3 (v/v) for 30 min each, ending with pure xylene for 30 min.
  4. Continue with mixtures of xylene and paraplast plus at the ratios 3:1, 1:1 and 1:3 (v/v) for 30 min each at 42 °C.
  5. Immerse the samples into pure paraplast plus for 2-3 days and keep them in a laboratory drying chamber at 60 °C. Transfer the samples into freshly prepared paraplast plus twice a day.
  6. Embed the leaf midrib samples in paraplast plus blocks (Figure 2C).
  7. Cut leaf midrib cross-sections, 10-15 micrometer-thick, with a microtome.
  8. Transfer the cross-sections to water droplets on circle glass slides coated with (3-aminopropyl)triethoxy-silane. After relaxation of the sections, carefully remove the water, place the slides on a slide warmer, and allow them to air dry for 2 h at 45 °C.
  9. Deparaffinize the cross-sections in two successive immersions in xylene for 3 min each, followed by rinsing 3 min each with mixtures of xylene and ethanol at the ratios 3:1, 1:1 and 1:3 (v/v), and complete the process with two successive rinses in 100% ethanol for 3 min each.
  10. Allow the cross-sections to air dry for 20 min and keep them in sterile Petri dishes.
  11. Determine the dimensions of tip cantilever from the calibrated light microscopy image, and insert the tip cantilever into the probe holder.
  12. Calibrate the deflection sensitivity of the tip cantilever on a clean fused silica surface using a PeakForce QNM in air mode. Use a single ramp test. Accept the value to be automatically entered into the deflection sensitivity parameter. (Fused silica is a suitable reference sample for determination of deflection sensitivity of stiff cantilevers, approximately 5 N/m, which are required for the measurement of samples with MOE less than 10 GPa.)
  13. Use the Nanoscope Thermal Tune function with a simple harmonic oscillator model to obtain the quality factor (Q value) and the resonance frequency (v0 value) of the tip cantilever. Following the Sader’s method of spring constant calibration, enter the dimensions of tip cantilever together with the above values for the quality factor and the resonance frequency, and directly calculate the stiffness of the tip cantilever on this website: http://www.ampc.ms.unimelb.edu.au/afm/calibration.html. Add the calculated value into the spring constant parameter window.
  14. Image one peak of TGT1 tip grating using a PeakForce QNM, and calculate the tip radius for expected depth. Use the fact that the tip might be approximated by ellipsoid surface. Calculate the fitted ellipsoid below the expected depth using the least square method. If the radii are similar, use the average. In other cases, use another tip or anticipate a non-ideal tip.
  15. In case of difficulties with the calibration of deflection sensitivity, spring constant or tip radius for a novice user, consult the calibraton chapter of the PeakForce QNM User Guide (available on the websites: http://nanopicolab.cnsi.ucla.edu/pages/publicview/manuals/PEAKFORCE_QNM_USERS_GUIDE-A.pdf or http://www.torontomicrofluidics.ca/cms/manuals/peak_force.pdf).
  16. Place the circle glass slide containing the sample cross-section onto a steel sample mounting disk using an adhesive pad.
  17. Place the steel sample mounting disk with the glass slide onto the scanner tube of a microscope.
  18. Image the tracheary element cell wall fragment within the primary xylem tissue (Figure 2D) by adjusting the peak force setpoint to achieve a sufficient sample deformation of 2 nm.
  19. Perform the measurement of cell wall topography (i.e. heights), MOE, adhesion, deformation, and energy dissipation at low approach tip velocities (approximately 0.3-0.5 μm/s).
  20. Import the raw data of MOE into the MATLAB software. The data of MOE must be filtered in order to account for the slippery effect of the tip on the steep cell wall surface. Calculate the height gradients (by means of a function “gradient”) using the height data of surface topography, and also calculate the surface slope (an angle between the gradient vector and the horizontal x, y plane) for each image pixel. Disregard MOE values where the surface slope exceeds the value of 30 degrees, and remove these values from the quantification of MOE. The values for other nanomechanical properties such as adhesion, deformation, and energy dissipation do not seem to be effected by high surface slope. However, to be consistent with the spatial distribution and the statistics of the measured characteristics, we suggest that the filtered data of adhesion, deformation, and energy dissipation be taken into account as well.


    Figure 2. Leaf midrib separation and preparation of a primary xylem tissue for the PeakForce QNM measurement. A. Fully expanded leaf of Ulmus, free of leaf midrib damage. The red boxes indicate the sites of leaf midrib separation. Scale bar = 1 cm. B. Leaf midrib samples separated for the tissue fixation and embedding. Scale bar = 1 cm. C. Leaf midrib sample embedded in a paraplast plus block, ready for microtome sectioning. Scale bar = 1 cm. D. Primary xylem tissue and lignin autofluorescence within tracheary elemet cell walls. The red box indicates the cell wall fragment subjected to the PeakForce QNM measurement. Scale bar = 100 μm

Representative data



Figure 3. PeakForce QNM images of cell wall fragment surfaces of leaf midrib tracheary elements in the Dutch elm disease-tolerant hybrid 'Dodoens' infected by the pathogenic fungus Ophiostoma novo-ulmi ssp. americana × novo-ulmi. A. Quantitative image for flatten height. B. Quantitative image for modulus of elasticity. C. Quantitative image for adhesion. Scale bars = 1.6 μm


Figure 4. PeakForce QNM images of cell wall fragment surfaces of leaf midrib tracheary elements in the natural hybrid species, Sorbus zuzanae. A. Quantitative image for flatten height. B. Quantitative image for modulus of elasticity. C. Quantitative image for adhesion. Scale bars = 1.1 μm

Recipes

  1. 0.1 M cacodylate buffer (pH 7.2, 500 ml)
    Dissolve 10.7 g sodium cacodylate trihydrate in 400 ml of distilled water
    Adjust the pH to 7.2 with HCl
    Add distilled water to make a total volume of 500 ml
    Handle in the fume cupboard
    Stored at room temperature
  2. Circle glass slides coated with (3-aminopropyl)triethoxy-silane
    Dip circle glass slides into acetone for 10 min
    Dry in a laboratory drying chamber
    Dip slides into 3% (v/v) (3-aminopropyl)triethoxy-silane in acetone for 15 min
    Rinse thoroughly in acetone
    Dry slides in a laboratory drying chamber
    Activate (3-aminopropyl)triethoxy-silane by placing slides into 2.5% (v/v) glutaraldehyde (Grade II) in 1x phosphate buffered saline for 1 h
    Wash slides in ultrapure water and air dry
  3. 1x phosphate buffered saline (1 L)
    Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in 800 ml of distilled water
    Adjust pH to 7.4 with HCl
    Add distilled water to make a total volume of 1 L
    Stored at room temperature
  4. Glutaraldehyde fixative solution [5% (v/v)]
    Prepared by diluting of 25% glutaraldehyde, Grade I in a 0.1 M cacodylate buffer
    Work with glutaraldehyde only in a properly operating and certified fume cupboard

Acknowledgments

The authors thank Dr. Olivier Arnould for sharing his MATLAB script for the calculation of tip radius from the TGT1 tip grating images, and Dr. Ingrid Čaňová for her technical assistance. This work was funded by the Slovak scientific grant agency VEGA (1/0132/12). The procedure described above was originally described in Ďurkovič et al. (2013).

References

  1. Ďurkovič, J., Čaňová, I., Lagaňa, R., Kučerová, V., Moravčík, M., Priwitzer, T., Urban, J., Dvořák, M. and Krajňáková, J. (2013). Leaf trait dissimilarities between Dutch elm hybrids with a contrasting tolerance to Dutch elm disease. Ann Bot 111(2): 215-227.
  2. Ďurkovič, J., Kardošová, M., Čaňová, I., Lagaňa, R., Priwitzer, T., Chorvát, D., Jr., Cicák, A. and Pichler, V. (2012). Leaf traits in parental and hybrid species of Sorbus (Rosaceae). Am J Bot 99(9): 1489-1500.
  3. Heu, C., Berquand, A., Elie-Caille, C. and Nicod, L. (2012). Glyphosate-induced stiffening of HaCaT keratinocytes, a Peak Force Tapping study on living cells. J Struct Biol 178(1): 1-7.
  4. Sweers, K., van der Werf, K., Bennink, M. and Subramaniam, V. (2011). Nanomechanical properties of alpha-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy, and Peakforce QNM. Nanoscale Res Lett 6(1): 270.

简介

称为PeakForce定量纳米机械映射(PeakForce QNM)的原子力显微镜(AFM)技术是用于纤维聚集体,人表皮细胞和木本植物细胞壁形貌的定量机械生物学成像的有效工具(Sweers等人, 2011; Heu等人,2012;Ďurkovič等人,2012;Ďurkovič等人,2013年)。在这里,我们描述一个详细的协议,用于测量木本植物中主要木质部细胞壁的纳米机械性能,用于确定降低的杨氏弹性模量(MOE),粘附,变形和能量耗散(图1)。这种新技术提供了对细胞壁样品中的最大负载力和变形深度的直接控制,保持压痕小,同时消除损伤侧向力,以便保持AFM尖端和植物样品。高分辨率和非破坏性成像为木质植物细胞壁的结构生物学提供了新的定量机械的见解。该程序还可适用于具有变化的刚度范围的其他生物样品。

关键字:原子力显微镜, 细胞壁力学, 弹性模量, 粘附, PeakForce QNM


图1.说明如何从力曲线提取不同的纳米力学性质的基本原理。该图显示了荷兰榆树病害敏感杂种"Groeneveld"中主要木质部细胞壁样品的典型力曲线, 。 PeakForce QNM模式对图像中的每个像素执行非常快的力曲线。力曲线数据的分析在飞行中进行,提供具有与高度图像相同分辨率的多个纳米机械性质的图。粘附力是基线和回缩曲线的最低部分之间的垂直距离。变形是接触点和转折点之间的水平距离(表示最大压痕)。杨氏弹性模量可以通过使用Derjaguin,Muller,Toporov(DMT)模型拟合在接触点后外推回缩曲线的线性部分来提取。根据最小二乘法方法程序确定线性部分的斜率。能量耗散可以通过对两条曲线之间的面积积分来计算。

材料和试剂

  1. 阔叶树(榆树 spp。, Sorbus spp。)
  2. 戊二醛溶液,25%的H 2 O(II级)(Sigma-Aldrich,目录号:G6257)
  3. 乙醇溶液(分析试剂)
    注意:它们各自通过在0.1M二甲胂酸盐缓冲液中稀释96%乙醇制备的10%,20%,30%,40%,50%,60%,70%,80%,90% 100%乙醇。
  4. 25%戊二醛(I级)(Sigma-Aldrich,目录号:G5882)
  5. 二甲苯(分析试剂)或具有较低毒性的替代性清洁剂(例如D-柠檬烯) 注意:只能在正常运行和认证的通风柜中使用二甲苯或D-柠檬烯。
  6. Paraplast plus(Sigma-Aldrich,目录号:P3683)
  7. (3-氨基丙基)三乙氧基硅烷(Sigma-Aldrich,目录号:A3648)
  8. 0.1 M二甲砷酸盐缓冲液(见配方)
  9. 用(3-氨基丙基)三乙氧基硅烷(参见配方)涂覆的圆形玻璃载玻片
  10. 1×磷酸盐缓冲盐水(PBS)(见Recipes)
  11. 戊二醛固定溶液(参见配方)

设备

  1. 剃刀刀片(细镊子和加热镊子)
  2. pH计
  3. 带真空泵的除油器
  4. 培养皿
  5. 实验室干燥室
  6. 收回基本雪橇切片机(8000系列)(Bright Instrument Co.)
  7. 圆形玻璃载玻片(直径11mm,1mm厚)
  8. 幻灯片
  9. 钢AFM样品安装盘
  10. 样品胶垫
  11. 具有180°旋转尖端和弹簧常数至少5N/m(Bruker AFM探针)的硅探针MPP-12120(型号:TAP150A)
  12. 熔融石英参考样品(标称MOE 72.9 GPa)或任何其他MOE值高于70 GPa的参考样品
  13. TGT1商用尖端表征光栅(NT-MDT Co.)
  14. 具有Nanoscope V控制器和PeakForce定量纳米机械映射模式(Bruker Corporation)的多模式8原子力显微镜

软件

  1. NanoScope分析(版本1.40r2)(Bruker Corporation)
  2. MATLAB(版本7)(MathWorks)

程序

  1. 从供体植物分离叶中叶样品(0.3-0.4cm×0.3-0.4cm)(图2A-B)。
  2. 将样品浸入5%(v/v)戊二醛(I级)在pH 7.2的0.1M二甲砷酸盐缓冲液中的固定溶液中,然后将其在室温下真空15分钟以从叶组织中除去空气。在4℃下将样品固定在固定溶液中6小时
  3. 在室温下,在升序的乙醇溶液系列中将样品脱水20分钟,接着以3:1,1:1和1:3(v/v)的比例分别使用乙醇和二甲苯的混合物30分钟,以纯二甲苯结束30分钟
  4. 继续以比例3:1,1:1和1:3(v/v)的二甲苯和paraplast plus的混合物在42℃下每次30分钟。
  5. 将样品浸入纯的paraplast加2-3天,并将其保存在60℃的实验室干燥室中。将样品转移到新鲜制备的paraplast中,每天两次。
  6. 将叶中叶样品嵌入paraplast加块(图2C)
  7. 用切片机切割10-15微米厚的叶中脉截面
  8. 将横截面转移到涂有(3-氨基丙基)三乙氧基硅烷的圆形玻璃载玻片上的水滴。松开切片后,小心取出水,将载玻片放在载玻片上,使其在45℃下风干2小时。
  9. 在二甲苯中连续浸渍两次,每次3分钟,然后用比例为3:1,1:1和1:3(v/v)的二甲苯和乙醇的混合物漂洗3分钟,将切片脱石蜡,该过程在100%乙醇中连续漂洗两次,每次3分钟。
  10. 让横截面风干20分钟,并保持在无菌培养皿
  11. 从校准的光学显微镜图像确定尖端悬臂的尺寸,并将尖端悬臂插入探头支架。
  12. 使用PeakForce QNM在空气模式下,在干净的熔融石英表面上校准尖端悬臂的偏转灵敏度。使用单斜坡测试。接受自动输入偏转灵敏度参数的值。 (熔融石英是确定刚性悬臂的偏转灵敏度的合适的参考样品,大约5N/m,这是测量MOE小于10GPa的样品所需的)。
  13. 使用具有简单谐波振荡器模型的Nanoscope热调谐功能来获得尖端悬臂的品质因数(Q值)和谐振频率(v0值)。按照Sader的弹簧常数校准方法,输入尖端悬臂的尺寸以及质量因子和共振频率的上述值,并直接计算该网站上尖端悬臂的刚度: http://www.ampc.ms.unimelb.edu.au/afm/calibration.html 。将计算的值添加到弹簧常数参数窗口中。
  14. 使用PeakForce QNM对TGT1尖端光栅的一个峰进行图像计算,并计算预期深度的尖端半径。使用尖端可能由椭圆体表面近似的事实。使用最小二乘法计算拟合的椭圆体低于预期深度。如果半径相似,请使用平均值。在其他情况下,使用另一个提示或预期不理想的提示
  15. 如果新手用户对偏转灵敏度,弹簧常数或尖端半径进行校准时遇到困难,请参阅PeakForce QNM用户指南的校准章节(可在网站上找到:http://nanopicolab.cnsi.ucla.edu/pages/publicview/manuals/PEAKFORCE_QNM_USERS_GUIDE-A.pdf http://www.torontomicrofluidics.ca/cms/manuals/peak_force.pdf )。
  16. 使用粘性垫将包含样品横截面的圆形玻璃载玻片放置在钢样品安装盘上
  17. 将钢样品安装盘与玻璃载玻片放在显微镜的扫描管上。
  18. 通过调整峰值力设定点以实现2nm的足够样品变形,在主要木质部组织内图像气管元件细胞壁碎片(图2D)。
  19. 在低接近尖端速度(约0.3-0.5μm/s)下进行细胞壁形貌(即高度),MOE,粘附,变形和能量耗散的测量。
  20. 将MOE的原始数据导入MATLAB软件。必须过滤MOE的数据,以便考虑尖端在陡峭的细胞壁表面上的光滑效应。使用表面形貌的高度数据计算高度梯度(通过函数"梯度"),并且还计算每个图像像素的表面斜率(梯度矢量和水平x,y平面之间的角度)。忽略表面坡度超过30度的值的MOE值,并从MOE的定量中除去这些值。其他纳米机械性能例如粘附性,变形和能量耗散的值似乎不受高表面斜率的影响。然而,为了与空间分布和测量的特性的统计一致,我们建议考虑粘附,变形和能量耗散的过滤数据。


    图2.叶中叶分离和用于PeakForce QNM测量的初级木质部组织的制备。A.榆木完全膨胀的叶,没有叶中叶损伤。红色框表示叶中脉分离的位点。比例尺= 1cm。 B.叶中叶样品分离用于组织固定和包埋。比例尺= 1cm。 C.叶片中脉样品嵌入paraplast加块,准备切片机切片。比例尺= 1cm。 D.主要木质部组织和木质素自体荧光在气管细胞壁内。红色框表示经过PeakForce QNM测量的细胞壁片段。比例尺=100μm

代表数据



图3.在病原真菌感染的荷兰榆树耐病杂种'Dodoens'中的叶中脉气管元件的细胞壁片段表面的PeakForce QNM图像 Ophiostoma novo-ulmi /strong> strong> 。A.定位图像,用于展平高度。 B.弹性模量的定量图像。 C.粘附的定量图像。比例尺=1.6μm


图4.天然杂交物种中叶中叶气管元件的细胞壁片段表面的PeakForce QNM图像 山梨 /strong> A.平整高度的定量图像。 B.弹性模量的定量图像。 C.粘附的定量图像。比例尺=1.1μm

食谱

  1. 0.1M二甲胂酸盐缓冲液(pH 7.2,500ml) 将10.7g二甲胂酸钠三水合物溶解在400ml蒸馏水中
    用HCl
    调节pH至7.2 加入蒸馏水,使总体积为500ml
    在通风橱中处理
    在室温下贮存
  2. 涂有(3-氨基丙基)三乙氧基硅烷的圆形玻璃载玻片
    浸入圆玻璃在丙酮中滑动10分钟
    在实验室干燥室中干燥
    浸入3%(v/v)(3-氨基丙基)三乙氧基硅烷在丙酮中15分钟 在丙酮中彻底冲洗
    在实验室干燥室中干燥载玻片
    通过将载玻片置于1×磷酸盐缓冲盐水中的2.5%(v/v)戊二醛(II级)中1小时来激活(3-氨基丙基)三乙氧基硅烷
    在超纯水中洗涤载玻片并风干
  3. 1×磷酸盐缓冲盐水(1L)
    将8g NaCl,0.2g KCl,1.44g Na 2 HPO 4,0.24g KH 2 PO 4,Na 2 HPO 4, 在800ml蒸馏水中的溶液 用HCl
    调节pH至7.4 加入蒸馏水使总体积为1 L
    在室温下贮存
  4. 戊二醛固定液[5%(v/v)]
    通过在0.1M二甲砷酸盐缓冲液中稀释25%戊二醛(I级)制备 仅在正确操作和认证的通风柜中使用戊二醛。

致谢

作者感谢Olivier Arnould博士分享他的MATLAB脚本用于计算TGT1尖端光栅图像的尖端半径,以及IngridČaňová博士的技术帮助。 这项工作由斯洛伐克科学资助机构VEGA(1/0132/12)资助。 上述程序最初描述于文献</em>中。 (2013年)。

参考文献

  1. Ďurkovič,J.,Čaňová,I.,Lagaňa,R.,Kučerová,V.,Moravčík,M.,Priwitzer,T.,Urban,J.,Dvořák,M. andKrajňáková,J.(2013)。 荷兰榆树杂交种与对荷兰榆树病具有对比耐受性的叶性状差异。 em> Ann Bot 111(2):215-227。
  2. Ďurkovič,J.,Kardošová,M.,Čaňová,I.,Lagaňa,R.,Priwitzer,T.,Chorvát,D.,Jr.,Cicák,A。和Pichler, Sorbus (蔷薇科)的亲本和杂交种的叶片性状。/a> 99(9):1489-1500。
  3. Heu,C.,Berquand,A.,Elie-Caille,C.and Nicod,L。(2012)。 草甘膦诱导的HaCaT角质形成细胞的硬化,对活细胞的峰力攻击研究。 178(1):1-7。
  4. Sweers,K.,van der Werf,K.,Bennink,M。和Subramaniam,V。(2011)。 α-突触核蛋白淀粉样蛋白原纤维的纳米机械性质:纳米压痕,谐波力显微镜和Peakforce的比较研究QNM。 Nanoscale Res Lett 6(1):270.
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引用:Ďurkovič, J., Kardošová, M. and Lagaňa, R. (2014). Imaging and Measurement of Nanomechanical Properties within Primary Xylem Cell Walls of Broadleaves. Bio-protocol 4(24): e1360. DOI: 10.21769/BioProtoc.1360.
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