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.
Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual
screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking".
However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on
an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this
limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our
approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid
suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation"
can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of
intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual
screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype
targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability).
Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse
paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including
lymphocytes, and cancer cells.
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