Principles and characteristics of multi-colour fluorescence imaging of plants

Green plants illuminated with UV-radiation emit a blue and green fluorescence as well as a red and far-red chlorophyll fluorescence with maxima near 440, 520, 690 and 740 nm, respectively. In contrast to point data measurements, the establishment of a high spatial resolution multi-colour fluorescence imaging system, the Karlsruhe/Strasbourg Fluorescence Imaging System (FIS), permits to simultaneously screen several hundred picture elements (pixels) of the leaf in the four fluorescence bands of plants, and opens new possibilities for physiology measurements and early stress detection. This review describes the principles and particular characteristics a) of the plants' fluorescence signatures, b) of fluorescence imaging in general and c) of the Karlsruhe/Strasbourg high resolution multi-colour fluorescence imaging system FIS. The origin of the fluorescence signals in the blue and green (cinnamic acids and other plant phenolics), the red and far-red (chlorophyll alpha) and the factors influencing these fluorescence signatures are explained. By means of an intensified video camera, fluorescence images with several hundred picture elements are taken, not only of the red and far-red Chi fluorescence, but also of the blue and green plant fluorescence, as multicolour images. Via computer-aided data processing one obtains false colour images not only of the fluorescence intensity, but also of the fluorescence ratios of whole leaves. The fluorescence ratios blue/red (F440/F630) and blue/far-red (F440/F740) proved to be very sensitive early stress and strain indicators of plants. The fluorescence ratio F690/F740 is an indicator of the in situ Chi content. By fluorescence imaging of the variable Chi fluorescence (Rfd-values) one can also simultaneously measure the potential photosynthetic activity of all points of a leaf. At short-term stress, the fluorescence ratio blue/green (F440/F520) is relatively stable, but changes at long-term stress, often by an increase of the green fluorescence emission. Due to the high statistic confidence (large pixel numbers) and the excellent possibilities to detect specific distribution gradients and local irregularities in fluorescence emission and fluorescence ratios over the leaf area, fluorescence imaging is a superior means for early stress and damage detection in plants.