Retrieving Spatiotemporal Information from Confocal Data: A Study Using Melanotrope Cells of Xenopus Laevis

Abstract

Confocal microscopy can be a useful tool for studies on cells in which local changes in intracellular ion concentrations need to be observed with a high spatial resolution. In time-related confocal studies high temporal resolution of changes in ion concentrations, detected as intensity changes of ion-specific fluorochromes like fura-red (Kurebayashi et al, 1993), can be achieved by collecting images at very short intervals (milliseconds). However, short collection periods inevitably introduce noise in the time traces, leading to difficulties in determining the starting time of changes in particular cell areas. If specimens are illuminated for prolonged periods, photobleaching becomes a very important concern. Since photobleaching is dependent on cell-specific anti-oxidant properties, it is virtually impossible to compare results obtained from different cells. In this communication we describe calculation methods carried out in experiments with isolated melanotropes of the amphibian Xenopus laevis, to determine changes in the intracellular concentrations of calcium using fura-red (Scheenen, 1995). Off line methods are presented to (1) correct for photobleaching and differences in loading characteristics between cells and (2) for removing high frequency noise, thus enabling the acquisition of accurate spatiotemporal data on fluorescence changes in different regions of a cell.