Fluorescence and luminescence techniques to probe ion activities in living plant cells.
M.D. Fricker1, C. Plieth1, H. Knight1, E. Blancaflor2, M.R. Knight1, N.S. White1 and
Simon Gilroy2
1Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.
2Biology Department, Pennsylvania State University, 208 Mueller Lab, University Park, PA 16803, USA.
Biology Department, 208 Mueller Laboratory, The Pennsylvania State University, University Park,
Pennsylvania 16802
Abstract.
Fluorescent probes offer almost unparalleled opportunities to visualize and quantify dynamic events within living cells, tissues or even organs with a minimum of perturbation. Although single cells or mono-layers maintained in culture can be readily imaged, measurements are often needed from cells within intact tissues. These cells are operating in their correct physiological context of cell-wall and cell-cell interactions. Imaging technology and dye-loading approaches have now progressed to where such in planta experiments are a real possibility. A range of different measurement techniques is now available to quantify fluorescence signals from reporter molecules within biological specimens. These include fluorimetry, flow cytometry, microscope photometry, camera, confocal and two-photon imaging. In addition, luminescence reporters, such as aequorin, are available to measure calcium using photomultiplier-based luminometer systems or photon-counting camera systems. Each of these approaches performs well for a specific range of measurement conditions and specimens. Thus, several techniques may need to be applied to provide a sufficiently flexible balance between the spatial, temporal and spectral resolution required to understand the physiological questions in the cell(s) of interest. Although the imaging and detector technology has rapidly advanced, two real problems remain. First, the techniques to introduce ion-selective probes into plant cells are not straightforward. Second, many of the reporter dyes do not behave predictably in the plant cell cytoplasm. Transgenic approaches using aequorin measurements have circumvented many of the problems associated with loading Ca2+ indicators. More recently, transgenic fluorescent calcium indicators have become available that exploit fluorescence resonance energy transfer (FRET) between diferent spectral forms of ågreen¼ fluorescent protein (Miyawaki et al., 1997). These probes offer great potential to combine the advantages of protein engineering and fluorescence techniques. Currently, however, the field of fluorescence and luminescence imaging of plant cell activities remains one fraught with potential artifacts and few generalized protocols. Most studies have required an extensive period of trial and error to determine what will, and will not, work with a particular cell type. Despite these limitations, perseverance has been rewarded by fascinating glimpses into cellular regulation. Coupling these imaging approaches to techniques such as caged probe technology has begun to allow us both to observe and to manipulate these cellular processes in the ultimate test-tube setting, the cell itself. The following sections provide guidelines on how to apply these approaches to plant specimens and how to identify, and hopefully avoid, many of the unique problems associated with quantitative fluorescence imaging and manipulation of plant cells.