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Journal of Biomedical Optics

Electron-multiplying charge-coupled detector-based bioluminescence recording of single-cell Ca2+
Author(s): Kelly L. Rogers; Jean-Rene Martin; Olivier Renaud; Eric Karplus; Marie-Anne Nicola; Marie Nguyen; Sandrine Picaud; Spencer L. Shorte; Philippe Brulet
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Paper 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.

Paper Details

Date Published: 1 May 2008
PDF: 10 pages
J. Biomed. Opt. 13(3) 031211 doi: 10.1117/1.2937236
Published in: Journal of Biomedical Optics Volume 13, Issue 3
Show Author Affiliations
Kelly L. Rogers, Institut Pasteur (France)
Jean-Rene Martin
Olivier Renaud, Institut Pasteur (France)
Eric Karplus
Marie-Anne Nicola, Institut Pasteur (France)
Marie Nguyen, Institut Pasteur (France)
Sandrine Picaud, Institut Pasteur (France)
Spencer L. Shorte, Institut Pasteur (France)
Philippe Brulet, Institut Pasteur (France)


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