Bioluminescence Imaging (BLI) is an increasingly useful and applicable technique that allows for the non-invasive
observation of biological events in intact living organisms, ranging from single cells to small rodents. Though the
photon production occurs within the host, significant exposure times can be necessary due to the low photon flux
compared to fluorescence imaging. The optical absorption spectrum of haemoglobin strongly overlaps most
bioluminescent emission spectra, greatly attenuating the total detectable photons in animal models. We have
developed and validated a technique that is able to red-shift the bioluminescent photons to the more desirable optical
region of > 650 nm, a region of minimal absorbance by hemoglobin. This red-shift occurs by using bioluminescence
as an internal light source capable of exciting a fluorophore, such as a fluorescent protein or a quantum dot, that
emits in the red. Interestingly, in the absence of an absorber, this excitation can occur over substantial distances
(microns to centimeters), far exceeding distances associated to, and thereby precluding, resonance energy transfer
phenomena. We show this novel technique yields a substantial increase in the number of red photons for in vitro and
ex vivo conditions, suggesting eventually utility for in vivo studies on, for example, intact living mice.
KEYWORDS: Video, Bioluminescence, Signal to noise ratio, Real time imaging, Imaging systems, CCD cameras, Signal detection, Infrared cameras, Cameras, Light sources and illumination
Bioluminescence imaging (BLI) allows detection of biological functions in genetically modified cells, bacteria, or animals expressing a luciferase (i.e., firefly, Renilla, or aequorin). Given the high sensitivity and minimal toxicity of BLI, in vivo studies on molecular events can be performed noninvasively in living rodents. To date, detection of bioluminescence in living animals has required long exposure times that are incompatible with studies on dynamic signaling pathways or nonanaesthetised freely moving animals. Here we develop an imaging system that allows: 1. bioluminescence to be recorded at a rate of 25 images/s using a third generation intensified charge-coupled device (CCD) camera running in a photon counting mode, and 2. coregistration of a video image from a second CCD camera under infrared lighting. The sensitivity of this instrument permits studies with subsecond temporal resolution in nonanaesthetized and unrestrained mice expressing firefly luciferase and imaging of calcium signaling in transgenic mice expressing green fluorescent protein (GFP) aequorin. This imaging system enables studies on signal transduction, tumor growth, gene expression, or infectious processes in nonanaesthetized and freely moving animals.
KEYWORDS: Calcium, Bioluminescence, Electron multiplying charge coupled devices, Sensors, Green fluorescent protein, Microscopes, Signal detection, Luminescence, Acquisition tracking and pointing, Video
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.
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