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.
Keywords: Chemical composition; Methods; Microspectroscopy; Multivariate analysis; Plant; Raman spectroscopy.