Among the first calcium indicators used for monitoring the dynamics of cellular calcium signaling were bioluminescent calcium-binding

signaling pathway photoproteins, such as aequorin (Ashley and Ridgway, 1968 and Shimomura et al., 1962). A next class of calcium indicators is represented by the synthetic compound arsenazo III, an absorbance dye that changes its absorption spectrum as a function of bound calcium (Brown et al., 1975). While aequorin and arsenazo III provided important early insights into the calcium-dependent regulation of neuronal processes (Hallett and Carbone, 1972, Llinás and Nicholson, 1975 and Stinnakre and Tauc, 1973), their implementation and use was often tedious, mostly because of problems with dye delivery. A true breakthrough was then the development of more sensitive and versatile fluorescent calcium PF-02341066 datasheet indicators and buffers by Roger Tsien and colleagues (Tsien, 1980). These indicators were the result of the hybridization of highly calcium-selective chelators like EGTA or BAPTA with a fluorescent chromophore. The first generation of fluorescent

calcium indicators consisted of quin-2, fura-2, indo-1, and fluo-3. Quin-2 is excited by ultraviolet light (339 nm) and was the first dye of this group to be used in biological experiments (Pozzan et al., 1982 and Tsien et al., 1982). Quin-2, however, is not particularly bright and needs to be used at high intracellular concentrations to overcome cellular autofluorescence (Tsien, 1989). Instead, another dye of that family, namely fura-2 (Grynkiewicz et al., 1985), is in many ways superior to quin-2 and became very popular among neuroscientists. Fura-2

is usually excited at 350 and/or 380 nm and shows calcium-dependent fluorescence Fossariinae changes that are significantly larger than the ones produced by quin-2. Furthermore, fura-2 is particularly useful because it allows more quantitative calcium measurements involving the ratioing of the signals obtained with alternating the excitation wavelengths (Neher, 1995). Over the years, many more calcium indicators with a wide range of excitation spectra and affinities for calcium have been introduced. These include, among others, the Oregon Green BAPTA and fluo-4 dye families (Paredes et al., 2008). These dyes are widely used in neuroscience because they are relatively easy to implement and provide large signal-to-noise ratios. An important next breakthrough, again from the laboratory of Roger Tsien (Miyawaki et al., 1997), was the introduction of protein-based genetically encoded calcium indicators (GECIs). While the early types of GECIs had somewhat limited areas of application because of their slow response kinetics and low signal-to-noise ratios, there had been tremendous progress in recent years (for review, see Looger and Griesbeck, 2011 and Rochefort et al., 2008).