Traditionally chlorophyll (Chl) and Chl precursors have been studied in vitro or in leaf tissue at low temperature. These methods are destructive and make it impossible to work with the same individual plant later on. In this paper we present a method for in vivo detection of Chl and its precursors in seedling plants which can be rescued for further studies. Multi-photon microscopy, which we show to be more reliable in vivo than UV-laser microscopy, is used to detect precursors in the biosynthetic pathway leading to Chl. The sensitivity and ability to distinguish different precursors with this system is compared to current methods. Furthermore, we report on optimization of the spectral scanning method with the aim to minimize the excitation light-evoked photo-conversion of the chlorophyll precursors.