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.

Presynaptic Ca2+ stores contribute to odor-induced responses in Drosophila olfactory receptor neurons

“…-sensitive bioluminescent photoprotein that can be utilized, in continuous recordings, for long-term imaging with an excellent signal-to-noise ratio (Martin et al, 2007;Baubet et al, 2000;Rogers et al, 2008). We have developed an in vivo preparation that allows the detection of odor-evoked changes in bioluminescence intensity in the sensory neuron terminals of a living fly at the level of the antennal lobes (Fig.1).…”

In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach

“…Since we were able to detect spontaneous activity in different parts and structures of the brain [19,37] we wondered whether Ca 2+ -activity could also be detected in the glial cells. In Drosophila, we used the repo-GAL4 driver, which allowed us to target all the glial cells.…”

Molecular Imaging of In Vivo Gene Expression

“…Transcriptional activity and second messenger signaling events can be visulaized in vivo because of the sensitivity of BRET [93]. Sensing Ca(II) ion concentration changes allows for the recording of spontaneous, transient events in real-time [93].…”

“…Sensing Ca(II) ion concentration changes allows for the recording of spontaneous, transient events in real-time [93]. Upon binding of Ca(II) to aqueorin with coelentrazine bound, BRET emission is recorded from the tethered GFP [93].…”

“…Sensing Ca(II) ion concentration changes allows for the recording of spontaneous, transient events in real-time [93]. Upon binding of Ca(II) to aqueorin with coelentrazine bound, BRET emission is recorded from the tethered GFP [93]. Transgenic fruit-flies expressing the Ca(II) BRET sensor in neurons were monitored over time for luminescence using an electron-multiplying CCD (EMCCD) [93].…”