Donohoe Bryon S, Ciesielski Peter N, Vinzant Todd B
National Renewable Energy Laboratory, Biosciences Center, Golden, CO, USA.
Methods Mol Biol. 2012;908:31-47. doi: 10.1007/978-1-61779-956-3_4.
Biomass exhibits structural and chemical complexity over multiple size scales, presenting many challenges to the effective characterization of these materials. The macroscopic nature of plants requires that some form of size reduction, such as dissection and microtomy, be performed to prepare samples and reveal features of interest for any microscopic and nanoscopic analyses. These size reduction techniques, particularly sectioning and microtomy, are complicated by the inherent porosity of plant tissue that often necessitates fixation and embedding in a supporting matrix to preserve structural integrity. The chemical structure of plant cell walls is vastly different from that of the membrane bound organelles and protein macromolecular complexes within the cytosol, which are the focus of many traditional transmission electron microscopy (TEM) investigations in structural biology; thus, staining procedures developed for the latter are not optimized for biomass. While the moisture content of biomass is dramatically reduced compared to the living plant tissue, the residual water is still problematic for microscopic techniques conducted under vacuum such as scanning electron microscopy (SEM). This requires that samples must be carefully dehydrated or that the instrument must be operated in an environmental mode to accommodate the presence of water. In this chapter we highlight tools and techniques that have been successfully used to address these challenges and present procedural details regarding the preparation of biomass samples that enable effective and accurate multi-scale microscopic analysis.
生物质在多个尺寸尺度上呈现出结构和化学复杂性，这给有效表征这些材料带来了诸多挑战。植物的宏观特性要求进行某种形式的尺寸减小，例如解剖和切片，以制备样品并揭示任何微观和纳米分析感兴趣的特征。这些尺寸减小技术，特别是切片和显微切片，因植物组织固有的孔隙率而变得复杂，这通常需要固定并嵌入支撑基质中以保持结构完整性。植物细胞壁的化学结构与细胞溶质中膜结合细胞器和蛋白质大分子复合物的化学结构有很大不同，而后者是结构生物学中许多传统透射电子显微镜（TEM）研究的重点；因此，为后者开发的染色程序对生物质并不适用。虽然与活植物组织相比，生物质的水分含量大幅降低，但残留水分对于在真空条件下进行的微观技术（如扫描电子显微镜（SEM））来说仍然是个问题。这就要求样品必须仔细脱水，或者仪器必须在环境模式下操作以适应水的存在。在本章中，我们重点介绍已成功用于应对这些挑战的工具和技术，并提供有关生物质样品制备的程序细节，这些程序能够实现有效且准确的多尺度微观分析。
