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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 Ca2+ requires the measurement and localization of changes in cytosolic free Ca2+, [Ca2+]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 Ca2+. When used with recently developed microscope technologies (e.g. fluorescence ratio imaging or confocal scanning laser microscopy), subcellular localization and precise quantification of [Ca2+]i dynamics in single cells can be achieved. This review describes the principles of [Ca2+]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 [Ca2+]i involving genetic transformation with the aequorin gene.

The production of endothelium-derived relaxing factor (EDRF), known to be nitric oxide (NO), is triggered by a rise in the cytoplasmic calcium concentration ([Ca2+]i) subsequent to receptor binding of vasoactive agonists. In vascular endothelial cells, NO is synthesized from L-arginine by the Ca2+/calmodulin-dependent NO synthase. In this study, we report the first simultaneous measurements of [Ca2+]i and [NO] at the level of single endothelial cells. In cultured bovine aortic endothelial cells, extracellular application of bradykinin (BK, 10 to 20 mumol/L) caused transient (sometimes oscillatory) increase in [Ca2+]i, which was measured with the fluorescent Ca2+ indicator fura 2 and fluorescence imaging microscopy. BK caused an increase in [Ca2+]i, primarily through release from intracellular stores. Under identical experimental conditions, BK caused a transient increase in [NO], which was measured by application of a porphyrinic NO microsensor. [NO] peaked at approximately 0.5 mumol/L. Simultaneous measurements of [Ca2+]i and [NO] in BK-stimulated endothelial cells revealed that a transient increase in [Ca2+]i was rapidly followed by an increase in [NO] that outlasted the [Ca2+]i transient.