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Pectin Nanostructure Visualization by Atomic Force Microscopy
采用原子力显微镜对果胶纳米结构进行可视化   

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Abstract

Pectins, complex polysaccharides rich in galacturonic acid, are a major component of primary plant cell walls. These macromolecules regulate cell wall porosity and intercellular adhesion, being important in the control of cell expansion and differentiation through their effect on the rheological properties of the cell wall. In fruits, pectin disassembly during ripening is one the main event leading to textural changes and softening. Changes in pectic polymer size, composition and structure have been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides but studies of detailed structure of isolated polymer chains are scarce (Paniagua et al., 2014). Atomic force microscopy (AFM) is a versatile and powerful technique able to analyze force measurements, as well as to visualize roughness of biological samples at nanoscale (Morris et al., 2010). Using this technique, recent research has found a close relationship between pectin nanostructural complexity and texture and postharvest behavior in several fruits (Liu and Cheng, 2011; Cybulska et al., 2014; Pose et al., 2015). Here, we describe an AFM procedure to topographically visualize pectic polymers from fruit cell wall extracts that has successfully been used in the study of strawberry ripening (Pose et al., 2012; Pose et al., 2015). Thus, from AFM images the 3D structural analysis of isolated chains (length, height, and branch pattern) can be resolved at high magnification and with minimal sample preparation. A full description of AFM fundamentals and the different sampling modes are described in Morris et al. (2010).

Keywords: Pectin(果胶), Nanostructure(纳米结构), AFM(原子力显微镜(AFM)), Strawberry(草莓), Cell wall(细胞壁)

Materials and Reagents

  1. Tri-distilled butanol (VWR International, catalog number: 20810.323 )
  2. Pectin fractions from cell wall extracts
    Notes:
    1. Cell wall extraction protocol is described in Posé et al. (2013).
    2. Pectin fractions from cell wall material are obtained by sequential extractions with CDTA buffer followed by sodium carbonate buffer, to solubilize a cell wall fraction enriched in ionically and covalently bound pectins respectively, as described in Posé et al. (2013) (see Recipes).
    3. Both pectin fractions (i.e., one extracted with CDTA and the other with sodium carbonate) were extensively dialyzed and stored until required at -20 ºC as aqueous solutions. Important: in order to prevent possible aggregation, any freeze-drying step must be avoided. It is recommended to aliquot the samples to avoid freeze-thawed successive cycles.
  3. Ammonium bicarbonate (FLUKA, catalog number: 09830 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 09830”.
  4. Trans-1,2-diaminocyclohexane-N,N,N'N'-tetra-acetic acid monohydrate (Sigma-Aldrich, catalog number: D 319945 )
  5. Sodium carbonate (Sigma-Aldrich, catalog number: S2127 )
  6. NaBH4 (Sigma-Aldrich, catalog number: D 452882 )
  7. 10 mM ammonium bicarbonate buffer (pH 8) (see Recipes)
  8. CDTA buffer (see Recipes)
  9. Sodium carbonate buffer (see Recipes)

Equipment

  1. Acoustic-isolated and temperature-controlled room (Figure 1A-1)
  2. Low-power light microscope, equipped with a television camera, was used to roughly position the AFM probe onto the top of the sample (Figure 1A-2)
  3. Atomic Force Microscope (East Coast Scientific Limited, Cambridge, UK) (Figure 1A-3; Figure 2A). Any AFM of suitable resolution can be used, although the details of the software and liquid cell will vary
  4. Anti-vibration table under the AFM microscope (Figure 1A-4)
  5. Photodiode amplifier (Figure 1A-5)
  6. Oscilloscope (Figure 1A-6; Figure 3)
  7. Digital control system composed by computer, DAC (digital analog converter) box, laser driver and high voltage amplifier. (for more details see Morris et al., 2010) (Figure 1A-7)
  8. PYREX® Culture Tubes with Rubber Liner Screw Caps (Thomas Scientific, catalog number: 9212C21 ) and teflon-lined caps
    Note: The screw-capped glass tubes were acid washed using a 1% hydrochloric acid solution overnight then rinsed with water.
  9. Sheets of mica (Elektron Technology, Agar Scientific, model: G250 ) cleaved with adhesive tape (3M, model: Magic Tape ) (Video 1)
  10. Short tip variety AFM probe model contact cantilevers (Budget Sensors SiNi, Bulgaria)
    Note: The tip is mounted on the edge of a V-shaped cantilever, the typical geometry used for topographical imaging (Figure 1B).
  11. Tip holder and open bucket liquid cell (Figure 1C; Vdeo 2)
  12. Basic equipment: pipettes, vortex, sonicator bath, heating block


    Figure 1. A. Photograph including an overview of AFM room set-up. The numeric labels are in accordance to the equipment list description. B. Scheme of a sharp tip located in the free end of a cantilever. C. Detail of tip holder (left) and liquid cell (right).

Software

  1. AFM software supplied with the instrument (SPM 6.01, ECS, Cambridge, UK)
  2. For the length measurements, images were converted to TIFF files using Paint Shop Pro v5.00 software (http://web.archive.org/web/19980514080113/http://jasc.com/)
  3. Image contrast and 3D effects were optimized using Gwyddion software v2.32
  4. AFM images were analyzed off-line using Image J v1.43u software (http://imagej.nih.gov/ij/index.html)
  5. Gwyddion is free and open source software, covered by GNU (General Public License) (http://gwyddion.net/)

Procedure

First preparative step, at the acoustic-isolated and temperature-controlled room, is to turn on air conditioning and all the AFM equipment (PC, monitor, amplifiers…) to avoid background noise during AFM scanning. In the meantime, you can prepare your samples as described next.

  1. Sample preparation
    1. Dilute pectin solution (pectin fractions obtained with CDTA and sodium carbonate buffer) in pure water to 1 mg/ml and heat at 80 °C for 30 min.
    2. Make a serial dilution in 10 mM ammonium bicarbonate* to a final concentration of 1-5 μg/ml. The required volume depends on the initial concentration of the sample taking into account that it would only 3 μl would be required for AFM. Dilutions must be prepared freshly just before used. A sublimable buffer is used to prevent deposition of buffer crystals on the mica substrate, because they don´t leave residual salt crystals when evaporates.
    3. Warm up the sample at 80 °C for 20 min and immerse in ultrasonic bath* for 10 min. *(only for sodium carbonate fraction; for CDTA fraction both dilution steps are done with pure water and the ultrasonic step is omitted). This step promotes disaggregation of cell wall networks to enable visualization of isolated chains.
    4. Reshape the mica to the final size, which will fit the liquid cell, and cleave the sheet inserting a sharp tip on the corner or peeling off the outer sheets with adhesive tape (Video 1). Always use tweezers to manipulate mica and keep them covered to protect from dust.

      Video 1. How to cleave the mica

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    5. Pipette out 3 μl of the CDTA or sodium carbonate pectin sample onto freshly cleaved mica and dry over heating stage at 37-40 °C for 20 min, in order to promote even spreading, to reduce molecular aggregation and to sublime off the buffer before imaging.
      Note: Incubation times, temperature and dilutions must be optimized for different polymers, always keeping in mind that those parameters must be consistent amongst samples that will be compared with each other.

  2. Atomic force microscopy
    1. Insert the sample into the liquid cell of the microscope (Figure 1C) and inject 300 μl of tri-distilled butanol into the cell, halfway of the sample approach sequence. The use of butanol as an imaging solvent has a double purpose; it eliminates capillary condensation but also is a precipitant for polysaccharides, so prevents desorbing of the polysaccharide during imaging.
    2. AFM tip probe is mounted on the tip holder (Figure 1B; Video 2), inserted into the microscope (Figure 1A-3) and roughly positioned on to the top of the sample using a low-power microscope equipped with a television camera (Figure 1A-2). The sharpness of the tip determines the resolving power of the instrument, so must be carefully manipulated as it can be blunted or contaminated with polysaccharide if driven into direct contact with the substrate.

      Video 2. How to mount sample on open bucket cell

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    3. Align laser and detector beams onto the tip using the four-quadrant photodiode and amplifier (Figure 2). In this case the tips used have a resonant frequency and force constant of 13 KHz and 0.4 N/m, respectively.
      1. Align the laser beam: The laser alignment knobs (represented with red arrows in Figure 2B) are used to move the laser in the X and Y directions until the laser spot is positioned on the end of the cantilever (Figure 2D). An optical microscope is used to visualise the laser spot and cantilever to check if the laser is aligned (Figure 2C-D)
      2. Align photodetector to the laser beam: Once the laser is aligned onto the cantilever, two different screws are used (represented with yellow arrows in Figure 2B) to position the photodiode so that it captures the reflected laser spot. Once it detects the reflected laser beam the photodiode must be finely adjusted to position the laser spot to a perfectly central position on the 4 quadrants of the photodiode. If the laser is not properly positioned on the cantilever it will be impossible to detect the reflected spot with the photodiode. The total voltage generated by the photodetector is shown in the electronic-combined box (Figure 2F). The level of cantilever deflection is shown in Figure 2E. This indicates the voltage difference between the top and bottom segments of the photodetector. For the contact mode, the deflection signal of the photodetector has to be close to zero.


        Figure 2. A. Atomic force microscope. B. AFM knobs to align the laser beam and photodetector. C. Probe with laser aligned. D. Probe with laser not aligned correctly. E. Photodetector amplifier with axis data. F. Photodetector amplifier with SUM data.

    4. Approach the sample to the tip to subtly position the tip onto the sample surface. This step must be done carefully to avoid hitting the tip onto the sample, which will blunt or contaminate the tip and ruin the scanned image. This step is done with the support of an oscilloscope (Figure 3) that reproduces the vibration of the cantilever. When the tip is far from the sample, the vibration waves will be broad (Figure 3A) and when the tip closely approaches the surface the wave pattern will a make sudden change denoting that tip and sample are establishing contact (Figure 3B). Adjust the force to obtain a good resolution during the scanning. Increasingly in modern AFMs this approach step is automatic.


      Figure 3. Oscilloscope output signals when tip and sample are at a far (A) or in correct (B) position. C. Scheme of different tip positions on the sample and its oscilloscope output signal. (see how those lines behave during the approaching sequence in Video 3)

      Video 3. A general overview of AFM method

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    5. Once everything is mounted and aligned, it is recommended to leave the sample and instruments 1 hour at 20 °C to equilibrate temperature and reduce background noise during scanning.
    6. The imaging is scanned at a scan speed of 2 Hz.
    7. AFM acquires data simultaneously from the movement of the feedback loop in the z-axis to keep a constant force during scanning (topographical mode image), and from the cantilever deflection (error signal image) with additional information about topography. Both topographical (Figure 4B) and error signal (Figure 4A) mode images are collected.
      Note: See Video 3 for a general overview of AFM method. Further information about alternative scanning modes and AFM in other biological samples are described in Morris et al. (2010).

  3. AFM image analysis
    1. Height measurements. The heights of features are analyzed using the AFM software supplied with the instrument (SPM 6.01, ECS, Cambridge, UK), which fits a plane and re-normalizes the AFM images. Topographical data is crucial for distinguishing true branch points from overlapping chains, where the height is doubled, and these are easily visualized as they appear as bright points at the crossing points on the chain (Figure 4).


      Figure 4. A. Representative image of CDTA pectins from ripe strawberry fruit obtained by AFM in contact mode. Branched pectin chains and micellar aggregates with emerging strands can be observed in the image. B. Height profiles, showing the heights in a true branch point (black arrow) of a polymer chain (profile 1) and micellar aggregates (profile 2) with emerging strands of same height than isolated chains (grey arrow) and higher height at the core area (arrowhead). Reprinted from Posé et al. (2015) with permission from Elsevier.

    2. Length measurements. The images are converted to TIFF files using Paint Shop Pro v 5.00 software. Image contrast and 3D layouts are performed by Gwyddion v 2.32 software. Then, the images are analyzed offline using ImageJ v1.43u software by plotting the strands with the freehand tool of the software. To determine the chain lengths only isolated chains, defined as individual strands that are not entangled with other strands, that are long enough to be exactly visualized, and which lay entirely within the scanned area are measured. The total length of the chain, including branches if any, is defined as the contour length.


      Figure 5. ImageJ workflow. A. First set the scale of your scan size (e.g., 256 pixels correspond to 1,000 nm). B. Chose freehand tool and draw the chains. C. Click CTRL+M and a new data window will prompt with the data measurements.

    3. Data analysis for length measurement.
      The contour length can be represented by histograms with the frequency of occurrence of particular lengths plot against the molecular length (Figure 6). Because polymers are mixtures of molecules of different molecular weight, they are naturally polydisperse, and their characterization is not well described with a single average. Instead, AFM data is characterized measuring the number-average () and the weight-average () contour length, as well as the ratio of the two average polymer lengths (), called polydispersity index (PDI). () is an arithmetic average (a), () is a weight average (b) that compensates the bias of a higher count of small chains over large ones (because is more likely to encounter small chains fully visualized in the scan area), and PDI a parameter to define the breadth of the distribution curve (Pose et al., 2012). This PDI index would be unity for a perfectly monodisperse polymer, so the higher the value the more disperse the polymer.

      An example of the intermediate calculations is included in table 1. Briefly, once contour length data is collected, interval Range should be defined (e.g., 0-25; 25-50; 50-100…), as well as the Frequency for each interval and its Mark Class (average interval range value). Then:
      Total (N) = Total number of data
      SUM1 = ∑ Mark Class x Frequency
      SUM2 = ∑ Mark Class x SUM1 
        = SUM1 / N
        = SUM2 / SUM1
      PDI =

      Table 1. AFM contour length analysis and intermediate calculations to obtain LN, LW and PDI


    4. Branching pattern. When true branch points are present, the number of branching points per isolated strand and their lengths can be measured. When equivalent dilutions are employed, the number of branched chains per scanned area can be used to define the percentage of branched polymers within a sample. Differences in the branching of the polymer chains, presence/absence of side chains and the number of side chains per backbone, are analyzed by a Chi-square test.
    5. Statistical distribution. Usually the frequency is a right skewed distribution which does not fit a normal distribution and median (ME) is the statistical parameter to analyze samples with Kruskal–Wallis test (Pose et al., 2012). Further statistical analysis of right skewed distributions data (Figure 6A-B) can be done when fit a Log normal distribution (Pose et al., 2012). Thus, if original data (L) is transformed by natural logarithm a normal distribution is obtained, and the data can be compared by analysis of variance (ANOVA). Another useful option for sample analysis is the cumulative frequency curves generated by the Log normal function (Figure 6C).


      Figure 6. Contour length distribution from CDTA (A) and sodium carbonate (B) soluble polymers. Bars represent observed data whilst the curved lines represent Log normal approximations. C. Cumulative frequencies for CDTA (black line) and Na2CO3 (grey line) pectic fractions by Log normal function normalized to the maximum peak obtained in each profile. N = 379 and 372 for CDTA and Na2CO3 samples, respectively. Reprinted from Posé et al. (2012) with permission from Elsevier.

      Note: It is recommended to analyze at least three dozen AFM images for each condition, with a minimum of 100 independent measurements of isolated chains from different images, to obtain a representative sample. Once everything is optimized, an AFM image is done every 5 min. See Video 3 for a general overview of AFM method.

Recipes

  1. 10 mM ammonium bicarbonate buffer (pH 8)
    Sterilized Millipore water filter is used.
    Either formic acid (HCOOH) or ammonium hydroxide (NH4OH) are recommended to adjust ammonium bicarbonate pH 8 buffer.
  2. CDTA buffer
    0.05 M trans-1,2-diaminocyclohexane-N,N,N'N'-tetraacetic acid in 0.05 M sodium acetate buffer (pH 6; adjust with Potassium Hydroxide KOH)
    Stored at RT
  3. Sodium carbonate buffer
    0.1 M sodium carbonate containing 0.1% NaBH4 freshly added
    Stored at RT but always add NaBH4 just before use
    Caution: NaBH4 is toxic, corrosive and dangerous when wet. Inhalation and contact with skin should be prevented.

Acknowledgments

Figure 4 is reprinted from Pose et al. (2015). Figure 6 is reprinted from Posé et al. (2012) with permission from Elsevier. This work was funded by the Ministerio de Educación y Ciencia of Spain and Feder EU Funds (grant reference: AGL2011-24814). The research at IFR was supported through the BBSRC core grant to the Institute.

References

  1. Liu, D. and Cheng, F. (2011). Advances in research on structural characterisation of agricultural products using atomic force microscopy. J Sci Food Agric 91(5): 783-788.
  2. Morris, V. J., Kirby, A. R. and Gunning, A. P. (2010). Atomic Force Microscopy for Biologists. 2nd ed. Imperial College Press ISBN-10: 184816467X.
  3. Paniagua, C., Pose, S., Morris, V. J., Kirby, A. R., Quesada, M. A. and Mercado, J. A. (2014). Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy. Ann Bot 114(6): 1375-1383.
  4. Posé, S., Kirby, A. R., Mercado, J. A., Morris, V. J. and Quesada, M. A. (2012). Structural characterization of cell wall pectin fractions in ripe strawberry fruits using AFM. Carb Pol 88: 882-890.
  5. Pose, S., Kirby, A. R., Paniagua, C., Waldron, K. W., Morris, V. J., Quesada, M. A. and Mercado, J. A. (2015). The nanostructural characterization of strawberry pectins in pectate lyase or polygalacturonase silenced fruits elucidates their role in softening. Carbohydr Polym 132: 134-145.
  6. Pose, S., Paniagua, C., Cifuentes, M., Blanco-Portales, R., Quesada, M. A. and Mercado, J. A. (2013). Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits. J Exp Bot 64(12): 3803-3815.
  7. Zdunek, A., Koziol, A., Pieczywek, P. M. and Cybulska, J. (2014). Evaluation of the nanostructure of pectin, hemicellulose and cellulose in the cell walls of pears of different texture and firmness. Food Bio Tech 7 (12): 3525-3535.

简介

果胶,富含半乳糖醛酸的复合多糖是主要植物细胞壁的主要成分。这些大分子调节细胞壁孔隙度和细胞间粘附,在通过它们对细胞壁的流变性质的影响来控制细胞扩增和分化中是重要的。在果实中,果胶在成熟期间的拆卸是导致质地变化和软化的主要事件。已经通过常规技术研究了果胶聚合物尺寸,组成和结构的变化,其中大多数依赖于多糖群体的批量分析,但是对分离的聚合物链的详细结构的研究很少(Paniagua等人, >,2014)。原子力显微镜(AFM)是一种通用且强大的技术,能够分析力测量,以及以纳米尺度可视化生物样品的粗糙度(Morris等人,2010)。使用这种技术,最近的研究发现果胶纳米结构复杂性和几种果实的纹理和收获后行为之间的密切关系(Liu和Cheng,2011; Cybulska等人,2014; Pose等。,2015)。在这里,我们描述了AFM程序,以在地形学上可视化来自果实细胞壁提取物的果胶聚合物,其已经成功地用于草莓成熟的研究中(Pose等人,2012; ,2015)。因此,从AFM图像,可以在高放大率和最小的样品制备下分离孤立链(长度,高度和分支模式)的3D结构分析。 AFM基本原理和不同采样模式的完整描述在Morris等人(2010)中描述。

关键字:果胶, 纳米结构, 原子力显微镜(AFM), 草莓, 细胞壁

材料和试剂

  1. 三蒸馏丁醇(VWR International,calalog number:20810.323)
  2. 来自细胞壁提取物的果胶级分
    注意:
    1. 细胞壁提取方案描述于Poséet al。 (2013)。
    2. 来自细胞壁材料的果胶级分通过顺序获得 用CDTA缓冲液,然后用碳酸钠缓冲液萃取 溶解富集离子和共价的细胞壁级分 如Posé等人,(2013)(参见Recipes)中所述。
    3. 两种果胶级分(即一种用CDTA提取 和另一种用碳酸钠)进行广泛透析 储存,直到需要在-20℃作为水溶液。 重要:在 为了防止可能的聚集,任何冷冻干燥步骤必须 避免。 建议将样品等分,以避免冻融   连续循环。
  3. 碳酸氢铵(FLUKA,目录号:09830)
    注意:目前,它是"Sigma-Aldrich,目录号:09830"。
  4. 反式-1,2-二氨基环己烷-N,N,N'N'-四乙酸一水合物(Sigma-Aldrich,目录号:D 319945)
  5. 碳酸钠(Sigma-Aldrich,目录号:S2127)
  6. NaBH 4(Sigma-Aldrich,目录号:D452882)
  7. 10mM碳酸氢铵缓冲液(pH8)(参见配方)
  8. CDTA缓冲区(参见配方)
  9. 碳酸钠缓冲液(见配方)

设备

  1. 隔音和温度控制室(图1A-1)
  2. 使用配备有电视摄像机的低功率光学显微镜将AFM探针粗略地放置在样品的顶部(图1A-2)。
  3. 原子力显微镜(East Coast Scientific Limited,Cambridge,UK)(图1A-3;图2A)。 可以使用任何合适分辨率的AFM,但软件和液晶盒的细节会有所不同
  4. 在AFM显微镜下的抗振台(图1A-4)
  5. 光电二极管放大器(图1A-5)
  6. 示波器(图1A-6;图3)
  7. 数字控制系统由计算机,DAC(数字模拟转换器)盒,激光驱动器和高压放大器组成。 (更多细节参见Morris等人,2010)(图1A-7)
  8. PYREX ®带橡胶衬垫螺帽的文化管(Thomas Scientific,目录号:9212C21)和特氟隆衬里的盖子
    注意:使用1%盐酸溶液将螺旋盖玻璃管酸洗过夜,然后用水冲洗。
  9. 用胶带(3M,型号:Magic Tape)切割的云母片(Elektron Technology,Agar Scientific,型号:G250)(视频1)
  10. 短尖端种类AFM探针模型接触悬臂(Budget Sensors SiNi,保加利亚)
    注意:尖端安装在V形悬臂的边缘,这是用于形貌成像的典型几何形状(图1B)。
  11. 尖嘴架和开式液桶(图1-C; Vdeo 2)
  12. 基本设备:移液器,涡流,超声波浴,加热块


    图1。 A。 照片包括AFM室设置的概述。数字标签与设备列表说明一致。 B.位于悬臂的自由端的尖端的方案。 C.尖端保持器(左)和液体电池(右)的细节。

软件

  1. AFM软件(SPM 6.01,ECS,Cambridge,UK)
  2. 对于长度测量,使用Paint Shop Pro v5.00软件( http://web.archive.org/web/19980514080113/http://jasc.com/
  3. 使用Gwyddion软件v2.32
    优化图像对比度和3D效果
  4. 使用Image J v1.43u软件( http://imagej.nih。)离线分析AFM图像。 gov/ij/index.html
  5. Gwyddion是免费的开源软件,由GNU(通用公共许可证)提供( http://gwyddion.net/

程序

在声学隔离和温度控制室处的第一制备步骤是在AFM扫描期间打开空调和所有AFM设备(PC,监视器,放大器...)以避免背景噪声。 在此期间,您可以按照下面所述准备样品。

  1. 样品准备
    1. 稀释果胶溶液(用CDTA和钠获得的果胶级分 碳酸盐缓冲液)纯化至1mg/ml,并在80℃加热30分钟。
    2. 在10mM碳酸氢铵中连续稀释至终浓度 浓度为1-5μg/ml。所需的音量取决于初始音量 考虑到样品的浓度只有3微升 将需要AFM。稀释液必须刚刚准备好 使用前。可升华的缓冲液用于防止缓冲液的沉积  晶体在云母基底上,因为它们不会留下残留的盐 蒸发时的晶体
    3. 将样品在80℃下加热20分钟  浸入超声波浴中10分钟。 * (仅适用于碳酸钠 分数;对于CDTA级分,两个稀释步骤用纯水进行  并省略超声步骤)。此步骤促进分解 的细胞壁网络,以实现孤立链的可视化
    4. 将云母重塑成最终尺寸,这将适合液体电池,和 切割片材在角上插入尖锐的尖端或剥离 外胶带(视频1)。总是  使用镊子操纵云母和保持它们覆盖,以保护 灰尘。

      视频1.如何切割云母
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    5. 吸出3微升的CDTA或碳酸钠果胶样品 新鲜切割的云母并在加热阶段在37-40℃干燥20分钟, 以促进均匀扩散,减少分子聚集 以在成像之前升华缓冲器。
      注意:孵化时间, 温度和稀释度必须针对不同的聚合物进行优化, 始终记住这些参数必须一致 将会彼此比较的样本。

  2. 原子力显微镜
    1. 将样品插入显微镜的液体细胞(图1C)和   注入300微升三蒸馏丁醇的细胞,中途 样本逼近序列。 使用丁醇作为成像溶剂具有   双重目的 它消除了毛细管冷凝,但也是一个 沉淀剂用于多糖,因此防止解吸 多糖。
    2. AFM尖端探头安装在尖端上 (图1B;视频2),插入 显微镜(图1A-3)和大致位于顶部的 样品使用配备有电视摄像机的低倍显微镜 (图1A-2)。 尖端的尖锐度决定分辨力 的仪器,因此必须仔细操纵,因为它可以钝   或如果与多糖直接接触则被污染 衬底。

      视频2. 如何在开放存储桶单元上挂载示例
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    3. 使用四象限将激光和检测器光束对准尖端 光电二极管和放大器(图2)。 在这种情况下,使用的提示有一个 谐振频率和力常数为13 KHz和0.4 N/m, 分别。
      1. 对准激光束:激光对准旋钮 (用图2B中的红色箭头表示)用于移动激光器   X和Y方向,直到激光光斑位于末端   悬臂(图2D)。 使用光学显微镜可视化 激光光斑和悬臂来检查激光是否对准(图 2C-D)
      2. 将光电探测器对准激光束:一旦激光器 对准到悬臂上,使用两个不同的螺钉(表示 与图2中的黄色箭头,B)以定位光电二极管,使其  捕获反射的激光光斑。一旦它检测到反射的激光 光束必须精细调整以定位激光光斑 到光电二极管的4个象限上的完全中心位置。如果  激光器不能正确地定位在悬臂上 不可能用光电二极管检测反射光斑。总数 由光电检测器产生的电压示于图中 电子组合盒(图2F)。悬臂偏转的水平 如图2E所示。这表示之间的电压差  光电探测器的顶部和底部。对于接触模式,  光电检测器的偏转信号必须接近零

        图2。 A.原子力显微镜。 B. AFM旋钮对准激光 光束和光电探测器。 C.探头与激光对准。 D.探头与激光   未正确对齐。 E.带轴数据的光电探测器放大器。 F。 带有SUM数据的光电探测器放大器。

    4. 接近样品 尖端将尖端巧妙地定位在样品表面上。 这一步 必须小心地进行,以避免将尖端撞到样品上 将钝化或污染尖端并毁坏扫描的图像。 这一步 是在再现示波器(图3)的支持下完成的 悬臂的振动。 当尖端远离样品时, 振动波将是宽的(图3A)和尖端紧密 接近表面波模式将使突然改变 表示尖端和样品建立接触(图3B)。 调整力以在扫描期间获得良好的分辨率。 在现代AFM中越来越多地采用这种方法步骤。


      图 3.示波器输出信号,当尖端和样品在远(A)或 在正确(B)位置。 C.不同提示位置的计划 示波器及其示波器输出信号。 (看看这些行的行为 在视频3)接近序列期间

      视频3. AFM方法的一般概述
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    5. 一旦一切安装和对齐,建议离开 样品和仪器在20℃下1小时以平衡温度和 减少扫描期间的背景噪音。
    6. 成像以2 Hz的扫描速度扫描。
    7. AFM从反馈回路的移动同时获取数据   在z轴上以在扫描期间保持恒定的力(形貌 模式图像),并且从悬臂偏转(误差信号图像) 有关地形的附加信息。 地形学(图   4B)和误差信号(图4A)模式图像。
      注意: 有关AFM方法的一般概述,请参阅视频3。 更多信息 关于其他生物样品中的替代扫描模式和AFM   描述在Morris et al。 (2010)。

  3. AFM图像分析
    1. 高度测量。 使用AFM分析特征的高度 软件(SPM 6.01,ECS,Cambridge,UK)提供, 其适合平面并且重新规格化AFM图像。 地形数据 是区分真正的分支点和重叠的关键 链,其中高度加倍,并且这些容易被视觉化为 它们在链条的交叉点处显示为亮点(图   4)。


      图4。 A.来自成熟的CDTA果胶的代表性图像 草莓果实通过AFM获得的接触模式。分支的果胶链  并可以观察到具有新兴链的胶束聚集体 图片。 B.高度剖面,显示真实分支点的高度 (黑色箭头)的聚合物链(轮廓1)和胶束聚集体 (轮廓2),其具有与分离链相同高度的新出现的链 (灰色箭头)和核心区域(箭头)处的较高高度。转载 来自Poséem et al。 (2015),获得Elsevier的许可。

    2. 长度 测量。使用Paint Shop将图像转换为TIFF文件 Pro v 5.00软件。图像对比度和3D布局通过执行 Gwyddion v 2.32软件。然后,使用离线分析图像 ImageJ v1.43u软件通过绘制的绳子与徒手工具  软件。为了确定链长仅为分离的链, 定义为不与其他链缠结的单独链,  它们足够长,可以完全可视化,并且完全可见 在测量区域内。 链的总长度, 包括分支(如果有的话),定义为轮廓长度

      图 5. ImageJ工作流程。 A.首先设置扫描大小的缩放比例(例如: 256像素对应于1000nm)。 B.选择徒手工具,绘制 链。 C.单击CTRL + M,新数据窗口将提示数据 测量。

    3. 长度测量的数据分析。
      的 轮廓长度可以由具有频率的直方图表示 出现特定长度与分子长度的关系 (图6)。 因为聚合物是不同分子的混合物 分子量,它们是自然多分散的,并且它们 表征没有很好地用单个平均值描述。 代替, AFM数据的特征在于测量数量平均值(), 重量平均()轮廓长度,以及 两个平均聚合物长度(),称为 多分散指数(PDI)。 ()是算术平均值(a),()是一个权重平均值(b),用于补偿较高计数的偏差 的小链在大的(因为更可能遭遇 小链在扫描区域完全可视化),并且PDI为参数 定义分布曲线的宽度(Pose 等人,2012)。这个 对于完全单分散的聚合物,PDI指数将是一致的,因此 该值越高,聚合物分散越多。

      中间计算的示例包括在表1中。 简而言之,一旦收集到轮廓长度数据,间隔 Range 应为  (例如:0-25; 25-50; 50-100 ...),以及每个的频率  间隔及其标记类(平均间隔范围值)。然后:
      总计(N)=总数据量
      SUM1 =Σ标记等级x频率
      SUM2 =ΣMark Class x SUM1 
        = SUM1/N
        = SUM2/SUM1
      PDI =

      表1. AFM轮廓长度分析和中间计算以获得L subN,L sub W和PDI 。

    4. 分支模式。当真分支点存在时, 可以测量每条分离链的分支点和它们的长度。 当使用等当量稀释时,支链的数目 每个扫描区域可以用于定义分支的百分比 聚合物。聚合物的支化差异 链,有无侧链和侧链的数目 每个骨架通过卡方检验分析
    5. 统计 分配。通常频率是一个右偏态分布 不适合正态分布,中值(ME)是统计 参数用Kruskal-Wallis检验分析样品(Pose等人 2012)。对右偏态分布数据的进一步统计分析 (图6A-B)可以在拟合Log正态分布时进行(Pose等人, al。,2012)。因此,如果原始数据(L)被自然变换 取对数正态分布,得到数据即可 通过方差分析(ANOVA)进行比较。另一个有用的选项 样本分析是由Log生成的累积频率曲线 正常功能(图6C)。


      图6.轮廓长度分布 从CDTA(A)和碳酸钠(B)可溶性聚合物。棒代表 观察数据,而曲线表示Log正态 近似。 CDTA的累积频率(黑线)和 通过对数正态函数归一化为(Na,Na)2 CO  在每个轮廓中获得的最大峰。对于CDTA,N = 379和372  分别为Na 2 CO 3和SO 3样品。转载自Posé等人(2012) Elsevier的许可

      注意:建议分析 每个条件至少三打AFM图像,最小为100 从不同图像的独立链的独立测量 获得代表性样品。 一旦一切都得到优化,AFM 图像每5分钟做一次。 有关AFM的一般概述,请参阅视频3 方法。

食谱

  1. 10mM碳酸氢铵缓冲液(pH8) 使用灭菌Millipore水过滤器。
    推荐使用甲酸(HCOOH)或氢氧化铵(NH 4 OH)调节碳酸氢铵pH 8缓冲液。
  2. CDTA缓冲区
    0.05M反式-1,2-二氨基环己烷-N,N,N',N'-四乙酸在0.05M乙酸钠缓冲液(pH 6;用氢氧化钾KOH调节) 存储在RT
  3. 碳酸钠缓冲液
    含有0.1%NaBH 4的0.1M碳酸钠新鲜加入
    存储在RT,但总是在使用前添加NaBH <4>
    警告:NaBH 4 是有毒,腐蚀性和潮湿的危险。 应避免吸入和接触皮肤。

致谢

图4从Pose (2015)转载。 图6在来自Elsevier的许可下从Posé等人(2012)重印。 这项工作由西班牙教育部长和Feder欧洲基金资助(资助参考:AGL2011-24814)。 IFR的研究得到了BBSRC对研究所的核心资助。

参考文献

  1. Liu,D。和Cheng,F。(2011)。 使用原子力显微镜对农产品结构表征的研究进展。 J Sci Food Agric 91(5):783-788。
  2. Morris,V.J.,Kirby,A.R.and Gunning,A.P。(2010)。原子力显微镜生物学家。第2版​​。帝国学院出版社,伦敦,ISBN-10:184816467X。
  3. Paniagua,C.,Pose,S.,Morris,V.J.,Kirby,A.R.,Quesada,M.A。和Mercado,J.A。(2014)。 水果软化和果胶分解:通过原子力显微镜评估纳米结构果胶修饰的概述。 114(6):1375-1383。
  4. Posé,S.,Kirby,A.R.,Mercado,J.A.,Morris,V.J.and Quesada,M.A。(2012)。 使用AFM在成熟草莓果实中对细胞壁果胶级分的结构表征。 Carb Pol 88:882-890。
  5. Pose,S.,Kirby,A.R.,Paniagua,C.,Waldron,K.W.,Morris,V.J.,Quesada,M.A。和Mercado,J.A。(2015)。 草莓果胶在果胶裂解酶或多聚半乳糖醛酸酶沉默果实中的纳米结构表征阐明了它们在软化中的作用。 a> Carbohydr Polym 132:134-145
  6. Pose,S.,Paniagua,C.,Cifuentes,M.,Blanco-Portales,R.,Quesada,M.A。和Mercado,J.A。(2013)。 了解多聚半乳糖醛酸酶FaPG1基因沉默对果胶基质拆解的影响,增强组织完整性和硬度成熟的草莓果实。 64(12):3803-3815。
  7. Zdunek,A.,Koziol,A.,Pieczywek,P.M。和Cybulska,J.(2014)。 评估梨果细胞壁中果胶,半纤维素和纤维素的纳米结构不同的质地和坚定度。 食物生物技术 7(12):3525-3535。
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引用:Posé, S., Paniagua, C., Kirby, A. R., Gunning, A. P., Morris, V. J., Quesada, M. A. and Mercado, J. A. (2015). Pectin Nanostructure Visualization by Atomic Force Microscopy. Bio-protocol 5(19): e1598. DOI: 10.21769/BioProtoc.1598.
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