| BIOLUMINISCENCE IMAGING OF SUBCELLULAR CALCIUM USING TARGETED AEQUORINS. |  |
Subcellular Ca2+ changes can be readly monitored by protein based, targeted probes as aequorin. However, single cell analysis has been hampered by the low level of light emission. This difficulty can be circumvected by high level expression of probes using herpex virus expression and high sensitivity photon counting cameras. For this end, cells expressing apoaequorin are incubated for 1-2 h at room temperature in standard medium with 1 µM coelenterazine. Then cells are placed into a incubation chamber thermostated to 37 oC mounted in a Zeiss Axiovert 100 TV microscope and perfused at 5-10 ml/min with the same medium with or without stimuli. At the end of each experiment, cells are permeabilized with 0.1 mM digitonin in 10 mM CaCl2 to release all the residual aequorin counts, a value required for calibration of the bioluminescence signal into subcellular Ca2+ concentrations. Bioluminescence images are taken with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor and integrated for 10 s periods. Photons/cell in each image were quantified using the Hamamatsu Aquacosmos software. Data are first quantified as rates of photoluminescence emission/total c.p.s remaining at each time and divided by the integration period (L/LTOTAL in s-1). Emission values of less than 4 c.p.s for individual cells are not used for calculations. More details about bioluminescence imaging of aequorin have been reported previously.
GH3 pituitary cells expressing aequorin targeted mitochondria display oscillations of mitochondrial calcium. Mitochondrial calcium oscillations are induced in silent cells after stimulation with thyrotropin releasing hormone (TRH) and depolarisation with high K medium.
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