Electron-multiplying charge-coupled detector-based bioluminescence recording of single-cell Ca2+

Abstract

The construction and application of genetically encoded intracellular calcium concentration ([Ca 2+ ] i ) indicators has a checkered history. Excitement raised over the creation of new probes is often followed by disappointment when it is found that the initial demonstrations of [Ca 2+ ] i sensing capability cannot be leveraged into real scientific advances. Recombinant apo-aequorin cloned from Aequorea victoria was the first Ca 2+ sensitive protein genetically targeted to subcellular compartments. In the jellyfish, bioluminescence resonance energy transfer (BRET) between Ca 2+ bound aequorin and green fluorescent protein (GFP) emits green light. Similarly, Ca 2+ sensitive bioluminescent reporters undergoing BRET have been constructed between aequorin and GFP, and more recently with other fluorescent protein variants. These hybrid proteins display red-shifted spectrums and have higher light intensities and stability compared to aequorin alone. We report BRET measurement of single-cell [Ca 2+ ] i based on the use of electron-multiplying charge-coupled-detector (EMCCD) imaging camera technology, mounted on either a bioluminescence or conventional microscope. Our results show for the first time how these new technologies make facile long-term monitoring of [Ca 2+ ] i at the single-cell level, obviating the need for expensive, fragile, and sophisticated equipment based on image-photon-detectors (IPD) that were until now the only technical recourse to dynamic BRET experiments of this type. © 2008 So ciety of Photo-Optical Instrumentation Engineers.