Figure 4.

Ca²⁺-induced bioluminescence was detected at the single-cell level. Neuro2A cells transfected with pGA (A.1–A.4) or pSG5A (B) were preincubated with 5 μM coelenterazine in a Ca²⁺-free buffer (A.3). GFP fluorescence made it possible to choose transfected cells. The background recorded before CaCl₂ addition (A.2) corresponds to the RLU level at time 0 of the experiment (A.4 and B). Intensities of fluorescence and bioluminescence activity are translated in scaled pseudocolors. Representative pictures of the chosen field are shown after addition of 5 mM CaCl₂ and 5 μM A23187 at 13 sec and 159 sec, respectively, after the beginning of the acquisition (A.1 and A.4). Each profile indicates the intensity of light emitted by a single cell. We defined five regions of interest by encircling individual cell soma. With pGA (data not shown) or pSG5A (B) transfection, a high concentration of CaCl₂ (100 mM) was added at the end of the experiment (500 sec) to determine whether the bioluminescent protein was still active (C). Control experiments were made with Fluo-3 AM on mock-transfected Neuro2A cells.