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植物学报 >

2024 , Vol. 59 >Issue 5: 783 - 791

DOI: https://doi.org/10.11983/CBB24063

技术方法

花粉管细胞壁原子力显微镜观测制样方法优化

  • 覃思颖 ,
  • 罗燕 ,
  • 张禾 ,
  • 胡君 ,
  • 廖菊够
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  • 1北京大学生命科学学院, 北京 100871
    2北京大学现代农学院, 北京 100871
    3云南大学生态与环境学院, 昆明 650500
*覃思颖, 北京大学生命科学学院公共仪器中心工程师, 负责原子力显微镜、流体力显微操作系统、共聚焦显微镜等大型仪器的技术支持与运行管理, 致力于多尺度生物样品的原子力显微镜观测、生物荧光成像与图像处理分析等技术服务, 为校内外100余个课题组提供技术支持, 支持用户在Nature、Cell、The Plant Cell等国际一流期刊发表论文70余篇, 已发表SCI论文6篇, 授权第一发明人专利1项, 2项技术与应用案例入选2023年中国仪器仪表学会科研仪器案例库。E-mail: qinsiying@pku.edu.cn

收稿日期: 2024-04-26

  录用日期: 2024-06-21

  网络出版日期: 2024-07-02

基金资助

国家自然科学基金(32060175);国家自然科学基金(32060043);云南省基础研究计划重点项目(202201AS070014)

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Optimization of Preparation and Detection Methods for Pollen Tube Cell Wall by Atomic Force Microscopy

  • Siying Qin ,
  • Yan Luo ,
  • He Zhang ,
  • Jun Hu ,
  • Jugou Liao
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  • 1School of Life Sciences, Peking University, Beijing 100871, China
    2School of Advanced Agricultural Sciences, Peking University, Beijing 100871, China
    3School of Ecology and Environmental Science, Yunnan University, Kunming 650500, China

Received date: 2024-04-26

  Accepted date: 2024-06-21

  Online published: 2024-07-02

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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

本文引用格式

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[J]. 植物学报, 2024 , 59(5) : 783 -791 . DOI: 10.11983/CBB24063

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

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