Microscopy Research and Technique

Biological systems have very different internal ion compositions in comparison with their surrounding media. The difference is maintained by transport mechanisms across the plasma membrane and by internal stores. On the plasma membrane, we can classify these mechanisms into three types, pumps, porters, and channels. Channels have been extensively studied, particularly since the advent of the patch clamp technique, which opened new windows into ion channel selectivity and dynamics. Pumps, particularly the plasma membrane Ca2+-ATPase, and porters are more illusive. The technique described in this paper, the self-referencing, ion-selective (or Seris) probe, has the ability to monitor the behavior of membrane transport mechanisms, such as the pumps and porters, in near to real-time by non-invasively measuring local extracellular ion gradients with high sensitivity and square micron spatial resolution.<br/><br/>The principles behind the self-referencing technique are described with an overview of systems utilizing ion, electrochemical and voltage sensors. Each of these sensors employs the simple expedient of increasing the system resolution by self-referencing and, thereby, removing the drift component inherent to all electrodes. The approach is described in detail, as is the manner in which differential voltage measurements can be converted into a flux value. For the calcium selective probes, we can resolve flux values in the low to sub pmol.cm-2s-1 range. Complications in the use of the liquid ion exchange cocktail are discussed. Applications of the calcium selective probe are given, drawing on examples from the plant sciences, developmental biology, muscle physiology, and the neurosciences.<br/>