Aequorins are excellent tools for measuring intra-organellar Ca2+ and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal. We also focus on bioluminescent Ca2+ imaging in nucleus or mitochondria of living cells. Finally, recent advances with a new chimeric GFP-aequorin protein (GAP), which can be used either as luminescent or fluorescent Ca2+ probe, are presented. GAP is robustly expressed in transgenic flies and mice, where it has proven to be a suitable Ca2+ indicator for monitoring physiological Ca2+ signaling ex vivo and in vivo.
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