Visualizing Calcium Signaling in Cells by Digitized Wide-Field and Confocal Fluorescent Microscopy

Calcium (Ca²⁺ ) is a fundamentally important component of cellular signal transduction. Dynamic changes in the concentration of Ca²⁺ ([Ca²⁺ ]) in the cytoplasm and within organelles are tightly controlled and regulate a diverse array of biological activities, including fertilization, cell division, gene expression, cellular metabolism, protein biosynthesis, secretion, muscle contraction, intercellular communication, and cell death. Measurement of intracellular [Ca²⁺ ] is essential to understanding the role of Ca²⁺ and for defining the underlying regulatory mechanisms in any cellular process. A broad range of synthetic and biosynthetic fluorescent Ca²⁺ sensors are available that enable the visualization and quantification of subcellular spatio-temporal [Ca²⁺ ] gradients. This chapter describes the application of wide-field digitized video fluorescence microfluorometry and confocal microscopy to quantitatively image Ca²⁺ in cells with high temporal and spatial resolution.