Imaging and measurement of cytosolic free calcium in plant and fungal cells

Abstract

SUMMARY Calcium plays a central role as a second messenger in plant and fungal cells and as such is involved in controlling numerous biological processes. Direct demonstration of signal‐response coupling via Ca 2+ requires the measurement and localization of changes in cytosolic free Ca 2+ , [Ca 2+ ] i , during these processes in living cells. In recent years this has become possible with the introduction of a range of fluorescent dyes (e.g. Indo‐1 Fura‐2 and Fluo‐3) which have a high affinity and selectivity for free Ca 2+ . When used with recently developed microscope technologies (e.g. fluorescence ratio imaging or confocal scanning laser microscopy), subcellular localization and precise quantification of [Ca 2+ ] i dynamics in single cells can be achieved. This review describes the principles of [Ca 2+ ] i imaging and measurement using fluorescent dyes, the equipment required to do it, the problems with botanical material and how they are being overcome, future developments for this approach in plant cell biology, and an entirely different strategy for the imaging and measurement of [Ca 2+ ] i involving genetic transformation with the aequorin gene.