Chapter 9: GFP Variants for Multispectral Imaging of Living Cells

Intercellular communication plays a coordinating role in the development of all multicellular organisms. This chapter uses the Arabidopsis thaliana root meristem as a model system for investigating intercellular interactions. Arabidopsis is amenable to genetic manipulation, and one can routinely generate transgenic lines for work with the intact organism. The chapter adapts the jellyfish green fluorescent protein (GFP) for use as a directly visible gene marker in Arabidopsis and develops genetic and optical techniques to visualize and manipulate cells within living plants. Unlike enzyme markers, GFP can be visualized at high resolution in living cells using confocal microscopy. The images are not prone to fixation or staining artifacts, and can be of exceptional clarity. Moreover, the activities of living cells, such as cytoplasmic streaming, are clearly evident during microscopy. Ordinarily, movement within a sample is a nuisance, placing constraints on the use of sometimes lengthy techniques for noise reduction during confocal microscopy, such as frame averaging. However, it is possible to monitor dynamic events by time-lapse confocal microscopy, and this combination of a vital fluorescent reporter with high-resolution optical techniques shows much promise for use in cell biological and physiological experiments.