Vim photon counting camera

Cells were transfected with the mitGA plasmid [25 (link)] using a Nucleofector II device (Amaxa Biosystems, Cologne, Germany) as reported previously [19 (link), 20 (link)]. This plasmids contain wild type aequorin targeted to mitochondria and a GFP sequence to select transfected neurons for bioluminescence imaging. After 24 h, cells were incubated for 2 h with 4 μM coelenterazine at room temperature. This incubation time is required for the reconstitution of aequorin enzyme, thus enabling Ca²⁺-dependent light emission. Then, cells were washed and placed into a perfusion chamber thermostated to 37°C under a Zeiss Axiovert S100 TV microscope. For bioluminescence imaging, cells were perfused at 5–10 ml/min with test solutions based on the standard perfusing solution described above prewarmed at 37°C. At the end of each experiment, cells were permeabilized with 0.1 mM digitonin in SEM containing 10 mM CaCl₂, added here to release all the residual aequorin counts. Bioluminescence images were taken with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor. Photonic emissions were integrated for 10 s periods. Photons were quantified using the Hamamatsu Aquacosmos software. Photonic emissions were converted to mitochondria free Ca²⁺ concentration ([Ca²⁺]mit) values as reported previously [19 (link), 20 (link), 32 (link)].