In this review, we take a retrospective look at the discovery and utilization of the Ca(2+) -sensitive bioluminescent protein complex, aequorin. We do consider the contribution it has made to our understanding of the natural phenomenon of bioluminescence, but it is in the application of extracted and purified aequorin as a reporter of Ca(2+) dynamics in living cells, which is arguably its major contribution to biological and biomedical science. Following its extraction, purification, and subsequent availability in the mid-1960s, aequorin became the intracellular reporter of choice until it was replaced in the late 1970s by easier-to-use fluorescence-based reporters. From the mid-1980s onwards, however, aequorin-based Ca(2+) imaging underwent a renaissance following the cloning of the aequorin gene and the emergence of routine techniques to target and express it exogenously in plant and animal systems. The development of aequorin as a tool continues as spectral varieties are being developed that allow simultaneous imaging of Ca(2+) dynamics in different cellular organelles and microdomains. We predict that further developments in the use of aequorin, as well as other bioluminescent proteins, will continue, especially in the areas of regenerative medicine and whole organism imaging.

In this chapter, we describe the practical aspects of measuring [Ca2+] transients that are generated in a particular cytoplasmic domain, or within a specific organelle or its periorganellar environment, using bioluminescent, genetically encoded and targeted Ca2+ reporters, especially those based on apoaequorin. We also list examples of the organisms, tissues, and cells that have been transfected with apoaequorin or an apoaequorin-BRET complex, as well as of the organelles and subcellular domains that have been specifically targeted with these bioluminescent Ca2+ reporters. In addition, we summarize the various techniques used to load the apoaequorin cofactor, coelenterazine, and its analogs into cells, tissues, and intact organisms, and we describe recent advances in the detection and imaging technologies that are currently being used to measure and visualize the luminescence generated by the aequorin-Ca2+ reaction within these various cytoplasmic domains and subcellular compartments.

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.

This chapter contains sections titled: Introduction Low Light Levels Methods of Coupling the Signal to the Detector Proximity Focusing Microscope Objectives Macro Lenses Fiber Optics Evaluating the Performance of an Optical System Numerical Aperture Transmission Efficiency Magnification Detector Technologies Selecting the Right Detector Detector Sensitivity Detector Noise Statistics of Photon Counting Summary References

Improved performance photodetectors are required for a variety of applications in Biomedical, Astronomy and High-Energy Physics research. The combination of high Quantum efficiencies, III–V photocathodes and Avalanche PhotoDiodes (APDs) can provide enhancements applicable to these research areas. This is particularly true in the photon counting regime. Preliminary development of a Winston-cone-based Hybrid device (ReFerence) and an APD-based imaging Hybrid Photomultiplier Tube are presented. These devices take advantage of the high-performance cathodes and mechanically robust structures used in state of the art night vision imagining systems. Theoretical and observed performance are described.

A microscope-based imaging system has been developed that records single photon emissions from luminescent samples. The time and position of each detected photon is recorded so that integration times for image reconstruction can be selected and adjusted during data analysis. The system performs automated acquisition of brightfield and epifluorescence images that are correlated with the photon images. The features of this system will be demonstrated in the context of studying the dynamics of living cells using luminescent markers such as luciferase and aequorin, which can be introduced by microinjection or transfection. The value of using this approach to study the effects of pharmacological agents on gene expression in living cells will be presented.