Exp Physiol 95.11 pp 1049–1060 Ca2+ events in living cells 1053 in what follows, but no attempt will be made to provide an overview of this vast ﬁeld. Bioluminescence and bioﬂuorescence: a Nobel calling Before going on to consider the new insights gained from Ca2+ imaging itself, it is worth mentioning the close conceptual and historical connections between the development of Ca2+ indicators and other live cell imaging techniques based on ﬂuorescent protein markers. In Shimomura and Johnson’s original work on Aequoria, they noticed that the jellyﬁsh produced a second optically active protein, which generated green ﬂuorescence when excited by the light emitted from the aequorin itself (Shimomura, 2009). At the time, this work was simply motivated by the researchers’ interest in the mechanisms responsible for light production in animals, and the discovery of green ﬂuorescent protein (GFP) in 1962 (Shimomura et al. 1962) remained of apparently limited interest for over 30 years. However, the cloning of the GFP gene in 1992 led to its expression in the nematode in 1994, the ﬁrst demonstration that it could be used as a marker of gene expression in living cells (Prasher et al. 1992; Chalﬁe et al. 1994). By introducing the DNA code for GFP linked to a gene of choice, the expression and trafﬁcking of any speciﬁed protein could now be visualized in living cells. Roger Tsien applied his understanding of ﬂuorescent chemistry, ﬁrst stimulated by the need to design improved Ca2+ indicators, to develop a range of modiﬁed ﬂuorescent proteins, allowing different gene products to be imaged simultaneously in the same organism or cell (Tsien, 2010). This work led to the rapid and diverse application of live cell imaging to address key questions in biology, a technical revolution for which Shimomura, Chalﬁe and Tsien were awarded the 2008 Nobel Prize for Chemistry (Chalﬁe, 2009; Shimomura, 2009; Zimmer, 2009; Tsien, 2010). Once again, new tools led to new knowledge. All of this came from Shimomura and Johnson’s fascination with creatures that glow. At the time, neither they nor anyone else could have guessed what amazing scientiﬁc and technological implications their ﬁndings would have. They just wanted to know! Perhaps this stands as a reminder that we need to leave room for curiosity-driven enquiry, no matter how little obvious application the work might have, since nobody can foretell where the next crucial ﬁnding will come from. If we could, it wouldn’t really be research. Confocal technology, Ca2+ imaging and Ca2+ sparks Getting back to the Ca2+ story, the step from Ca2+ measurement to Ca2+ imaging came with the development of new microscopy techniques. Recordings using Ca2+ indicators often involve a technique referred to as C 2010 The Author. Journal compilation C 2010 The Physiological Society microﬂuorimetry, in which the overall (i.e. spatially summed) intensity of any emitted ﬂuorescence is recorded. Although a microscope objective is used to collect the light, no ﬂuorescent image is captured. There are several technical reasons for doing this, including increased sensitivity and speed of recording, important with rapidly changing signals. However, the development of confocal microscopy and ever more sensitive CCD-based cameras made true Ca2+ imaging a viable option, in which local changes in [Ca2+]i within individual cells can be visualized. Confocal microscopes combine a scanning laser to excite cell ﬂuorescence with a pinhole to exclude out-of-focus light coming back into the imaging scanhead, allowing clear optical sections to be recorded from much thicker tissues. Initially, this technique was used to improve the resolution of images from ﬁxed specimens stained with ﬂuorescent dyes, and to generate three-dimensional tissue models by imaging different planes in the same sample and combining the data (Brakenhoff et al. 1989). It quickly became obvious, however, that this technology was also ideal for Ca2+ imaging using ﬂuorescent indicators in living cells (Niggli & Lederer, 1990). Rather than simply recording the average signal emitted from the sample, as is the case in Ca2+ microﬂuorimetry, it was now possible to repeatedly image the Ca2+-dependent ﬂuorescence in a given optical section of a cell or tissue, generating a ‘movie’ showing how Ca2+ changed over time in different cells and even in different parts of the same cell (Fig. 5). The only problem with these recordings was that they were too slow to faithfully reproduce the rapid changes in [Ca2+]i seen in muscle cells, which were the ﬁrst focus of interest. This limitation could be largely overcome by adapting the technique so that the laser did not scan across the whole of the image plane but repeatedly scanned back and forth along a single line. Temporal resolution was greatly increased (from about 1 frame s−1 to about 500 lines s−1), but at the cost of one spatial dimension. This sacriﬁce was soon shown to be justiﬁed, however, as the resulting linescan images revealed subcellular Ca2+ events previously unsuspected in cardiac muscle cells. These brief, spatially localized increases in [Ca2+]i were dubbed ‘Ca2+ sparks’ and were shown to represent spontaneous release of Ca2+ from the sarcoplasmic reticulum via cardiac ryanodine receptors (Cheng et al. 1993). It should also be noted in passing that localized spontaneous Ca2+- release events mediated by clusters of IP3Rs have also been identiﬁed in a range of excitable and non-excitable tissues and given the name ‘Ca2+ puffs’ (Smith et al. 2009; Wray & Burdyga, 2010). These will not be considered further. We will return to the function of Ca2+ sparks later, but ﬁrst I want to consider smooth muscle, which will be the main focus for the rest of this discussion. Interestingly, evidence that something similar to Ca2+ sparks must occur in smooth muscle had been reported nearly a decade before they were imaged. Electrophysiological recordings,