Optical microscopy has developed as an indispensable tool for Arabidopsis cell biology. This is due to the high sensitivity, good spatial resolution, minimal invasiveness, and availability of autofluorescent proteins, which can be specifically fused to a distinct protein of interest. In this chapter, we introduce the theoretical concepts of fluorescence emission necessary to accomplish quantitative and functional cell biology using optical microscopy. The main focus lies on spectroscopic techniques, which, in addition to intensity-based studies, provide functional insight into cellular processes.