Gorzsás András
Department of Chemistry, Umeå University, 90187, Umeå, Sweden.
Methods Mol Biol. 2017;1544:133-178. doi: 10.1007/978-1-4939-6722-3_12.
Raman microspectroscopic techniques provide highly detailed chemical information about xylem tissue at submicron spatial resolution. The techniques are generally sensitive and they provide a powerful, yet inexpensive way to probe the chemical composition of individual cells or cell wall layers in situ, non-destructively, in a confocal manner, simultaneously detecting all chemical compounds without the need of external agents (label, dyes, stains). Problems with limited specificity in complex chemical matrices such as cell walls may arise, compounded by fluorescence problems. However, these can often be circumvented. In this chapter, the basics of the technique, including a common instrumental setup, together with the general strengths and limitations of Raman microspectroscopy are discussed. Detailed protocols are provided for single point measurements, as well as for fully customizable raster scan mapping, including sample preparation, setup, and measurement steps. The major steps of the data analysis procedure are discussed as well.
拉曼显微光谱技术能够在亚微米空间分辨率下提供有关木质部组织的高度详细的化学信息。这些技术通常很灵敏，能提供一种强大且经济的方法，以共聚焦方式原位、无损地探测单个细胞或细胞壁层的化学成分，无需外部试剂（标记物、染料、染色剂）即可同时检测所有化合物。在诸如细胞壁这样的复杂化学基质中，可能会出现特异性有限的问题，荧光问题会使情况更加复杂。然而，这些问题通常可以规避。在本章中，将讨论该技术的基础知识，包括常见的仪器设置，以及拉曼显微光谱的一般优点和局限性。还提供了单点测量以及完全可定制的光栅扫描映射的详细方案，包括样品制备、设置和测量步骤。此外，还讨论了数据分析过程的主要步骤。
