title: Electron-multiplying charge-coupled detector-based bioluminescence recording of single-cell Ca2+.
abstract: The construction and application of genetically encoded intracellular calcium concentration ([Ca2+]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 [Ca2+]i sensing capability cannot be leveraged into real scientific advances. Recombinant apo-aequorin cloned from Aequorea victoria was the first Ca2+ sensitive protein genetically targeted to subcellular compartments. In the jellyfish, bioluminescence resonance energy transfer (BRET) between Ca2+ bound aequorin and green fluorescent protein (GFP) emits green light. Similarly, Ca2+ 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 [Ca2+]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 [Ca2+]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.
authors.affiliation: Plate-forme d'Imagerie Dynamique, Imagopole,Institut Pasteur, Paris, France. krogers@pasteur.fr
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Rogers
authors.forename: Jean-Rene
authors.initials: JR
authors.lastname: Martin
authors.forename: Olivier
authors.initials: O
authors.lastname: Renaud
authors.forename: Eric
authors.initials: E
authors.lastname: Karplus
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Nicola
authors.forename: Marie
authors.initials: M
authors.lastname: Nguyen
authors.forename: Sandrine
authors.initials: S
authors.lastname: Picaud
authors.forename: Spencer L
authors.initials: SL
authors.lastname: Shorte
authors.forename: Philippe
authors.initials: P
authors.lastname: Brûlet
mesh_terms: D000331:Aequorin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D004583:Electrons; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D008164:Luminescent Proteins / Q000378:metabolism*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D014159:Transducers
chemical_list: D008164:Luminescent Proteins; D000331:Aequorin; D002118:Calcium