Imaging techniques in plant transport: meeting review

This review covers recent advances in imaging techniques that were presente d during the 11th International Workshop on Plant Membrane Biology, Cambrid ge, August 1998. Cell biology has been revolutionized by the arrival of gre en fluorescent protein (GFP) and GFP is now routinely used as a cell-lineag e marker in plants, to localize proteins to subcellular compartments or as a tag to follow dynamics in endomembrane compartments. More recent developm ents include modification of GFP to form physiological sensors for calcium and pH. These provide a transgenic approach in parallel with conventional c hemical dyes to track signalling events or follow membrane recycling pathwa ys. Confocal microscopy has become a routine technique to visualize these f luorescent probes, particularly in intact tissues. Multi-photon microscopy may push the capability of imaging techniques further, allowing imaging at even greater depths and long-(red)-excitation of UV fluorochromes. The lumi nescent calcium indicator, aequorin, has been much more extensively used in plants than the transgenic fluorescent calcium indicators, and photon-coun ting imaging systems can now record calcium transients at video-rate in int act plants. Digital imaging, whether camera, confocal or multi-photon, prov ides quantitative data, but correct interpretation requires rigorous analys is of noisy and partially correlated imaging data. Statistical analysis by Bayesian inference may well become the most appropriate technique to handle such images, but has only recently been applied to ratio imaging in plants . In addition to generating images, light can also be used to manipulate in tracellular events using photolysis of caged probes. The control of the loc ation, timing and amplitude of the release allows exquisitely subtle manipu lation of signalling networks. More aggressive pulses of UV laser-light pro vide an equally powerful tool either to ablate whole cells completely for d evelopmental studies or to punch-out tiny holes in the cell wall to give ac cess to the plasma membrane for patch-clamping and electrophysiological inv estigation of cells in situ.