花粉管细胞壁原子力显微镜观测制样方法优化

doi:10.11983/CBB24063

摘要/Abstract

摘要： 原子力显微技术是研究植物细胞壁超微结构与力学性质的重要表征手段, 良好的样品制备是获得可靠数据的前提。花粉管作为微米级别的植物样品, 是研究细胞壁结构与功能的典型实验材料, 但是制样过程中活性生理状态下的花粉管与基底黏附不牢固, 难以获得活性生理状态下的花粉管原子力显微镜观测数据。该文以烟草(Nicotiana tabacum)花粉管为材料, 对花粉管原子力显微样品制备和观测方法进行优化, 采用固体培养基薄层作为花粉管与基底之间的黏附剂, 花粉管在萌发和生长的同时完成与基底的黏附。与常规的干燥-复水法相比, 优化的液下黏附法, 可在液体环境下直接观测, 无需经过干燥处理, 避免了干燥-复水过程对花粉管造成的形貌皱缩与力学性质改变, 能够获得活性生理状态下花粉管细胞壁原位的高分辨率原子力显微镜观测数据。该方法可应用于不同种属及不同尺寸花粉管的原子力显微镜观测。

关键词: 原子力显微镜, 花粉管, 细胞壁, 原位, 力学性质

Abstract: Atomic force microscopy (AFM) is a powerful tool for studying the ultrastructure and mechanical properties of plant cell walls, while good sample preparation is essential for AFM data acquisition. As micron-scale plant samples, pollen tubes are typical experimental materials for studying the structure and function of cell walls. Due to the difficulty in sample preparation, it is hard to obtain in situ AFM data in physiological state of pollen tubes as pollen tubes are hard to attach to the substrate firmly in fluid. In this study, the AFM preparation and detection methods are optimized using Nicotiana tabacum pollen tubes as the experimental materials. Thin solid medium is used as the adhesive, thereby pollen tubes are attached to the glass slide during germination and elongation in fluidic environment. Compared with the conventional drying-rehydration method, the optimized liquid-attaching method allows observation directly in fluid without drying treatment, which avoids the shrinkage and denaturation of pollen tubes caused by drying-rehydration process. Accordingly, liquid-attaching method can be applied for pollen tubes of different species and sizes, providing support to obtain high-resolution in situ AFM data of pollen tube cell walls in native physiological state under aqueous conditions.

Key words: atomic force microscope, pollen tube, cell wall, in situ, mechanical properties

中图分类号:

覃思颖, 罗燕, 张禾, 胡君, 廖菊够. 花粉管细胞壁原子力显微镜观测制样方法优化. 植物学报, 2024, 59(5): 783-791.

Siying Qin, Yan Luo, He Zhang, Jun Hu, Jugou Liao. Optimization of Preparation and Detection Methods for Pollen Tube Cell Wall by Atomic Force Microscopy. Chinese Bulletin of Botany, 2024, 59(5): 783-791.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24063

https://www.chinbullbotany.com/CN/Y2024/V59/I5/783

图/表 4

图1 花粉管原子力显微镜(AFM)制样及观测示意图 (A) 液下黏附法; (B) 干燥-复水法; (C) 载玻片待测区域(bar=1 cm); (D) AFM观测明场视野(bar=100 μm); (E) AFM液下观测

Figure 1 Schematic diagram of pollen tube atomic force microscope (AFM) preparation and detection (A) Liquid-attaching preparation method; (B) Drying-rehydration preparation method; (C) Test areas of glass slides (bar=1 cm); (D) Optical bright field image of AFM probe at work (bar=100 μm); (E) AFM detection under aqueous conditions

图2 干燥-复水法与液下黏附法的花粉管原子力显微镜(AFM)观测数据(顶端与中段部位) (A) 花粉管三维形貌图(颜色表示高度); (B) 叠加杨氏模量数据的花粉管三维形貌图(颜色表示杨氏模量)。Bars=3 μm

Figure 2 Pollen tube atomic force microscope (AFM) imaging data of drying-rehydration and liquid-attaching preparation methods (apical dome and distal region) (A) AFM-mapping of three-dimensional topography of pollen tubes (colors represent the height); (B) Three-dimensional topography of pollen tubes overlaid with Young’s modulus (colors represent the elasticity). Bars=3 μm

图3 不同黏附方法对花粉管顶端与中段杨氏模量的影响 (A)-(D) 干燥-复水法的花粉管顶端(A)、中段(C)与液下黏附法的花粉管顶端(B)、中段(D)的代表性力-距离曲线; (E) 花粉管顶端的杨氏模量图; (F) 花粉管中段的杨氏模量图(颜色表示杨氏模量, 虚线方框(边长3.5 μm)为统计杨氏模量平均值的区域); (G) 干燥-复水法与液下黏附法的花粉管顶端与中段的杨氏模量统计结果(***表示不同处理间在P<0.001水平差异极显著)。Bars=3 μm

Figure 3 Effects of different preparation methods on the Young’s modulus of pollen tube apical dome and distal region (A)-(D) Typical force-distance curves obtained from apical dome (A) and distal region (C) of drying-rehydration method, apical dome (B) and distal region (D) of liquid-attaching method; (E) Young’s modulus of pollen tube apical dome; (F) Young’s modulus of pollen tube distal region (colors represent the elasticity, dashed boxes (side length 3.5 μm) are the areas for calculating the average Young’s modulus); (G) The statistical results of Young’s modulus of pollen tube apical dome and distal region using drying-rehydration and liquid-attaching preparation methods (*** represent extremely significant differences among different treatments at P<0.001). Bars=3 μm

图4 不同黏附方法对花粉管顶端与中段截面高度的影响 (A), (D) 花粉管顶端(A)及中段(D)高度图(颜色表示高度, 虚线示截面位置); (B), (E) 不同黏附方法的花粉管顶端(B)及中段(E)截面高度图(箭头为截面高度差位置); (C), (F) 不同黏附方法的花粉管顶端(C)及中段(F)截面高度(Z)统计(***表示不同处理间在P<0.001水平差异极显著)。Bars=3 μm

Figure 4 Effects of different preparation methods on the cross-section height of pollen tube apical dome and distal region (A), (D) The height images of pollen tube apical dome (A) and distal region (D) (colors represent the height, and dashed lines represent the position of the cross-section); (B), (E) The cross-section height of pollen tube apical dome (B) and distal region (E) using different preparation methods (the arrows indicate the position of cross-section height); (C), (F) The statistical results of cross-section height (Z) of pollen tube apical dome (C) and distal region (F) using different preparation methods (*** represent extremely significant differences among different treatments at P<0.001). Bars=3 μm

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