Fluorescent Proteins - Introduction
Different fluorescent proteins can be found in natural organisms ranging from bacteria to insects or fish and in some cases they are responsible for bioluminescence. The emission of light can depend on a given conformation, the presence of a cofactor or the environment were the protein is placed (pH, ions, etc.).
Aequorea victoria, a jellyfish used in the 1960s to investigate bioluminescence, is one of those organisms capable of emitting light. Its bioluminescence is due to the presence of aequorin (sensitive to calcium) and green fluorescent protein (GFP, a fluorescent protein).
Aequorin has been used for calcium imaging during more than 30 years. It is a chemiluminescent protein: light is emitted on binding calcium, without the need of light irradiation. This makes the assay very sensitive but, due to the low signal, it requires long acquisitions, lowering the spatial and temporal resolution.
Although GFP is non-sensitive to calcium, it has been used as a basis for the development of many chimeras GFP-based fluorescent calcium indicators. The first derivatives were described in 1997 by R. Tsien et. al. and were based on a fluorescence resonance energy transfer (FRET) process. Since then, many other GFP-indicators have been described and most of them are based on calmodulin as calcium switch.
The main advantage of using fluorescent proteins in calcium quantification is the fact that recombinant proteins can be tagged to different organelles, so calcium can be measured specifically (i.e. endoplasmic reticulum, mitochondria, etc.). A drawback is that, in general, they are not commercially available.