Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca²⁺ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca²⁺ signaling derives from mechanisms by which Ca²⁺ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca²⁺ microdomains (Ca²⁺ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca²⁺ signals with a 128 x 128 pixel spatial resolution at rates of >500 frames sec^-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca²⁺ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP₃-mediated Ca²⁺ signals in intact mammalian cells loaded with Ca²⁺ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca²⁺ signals. The algorithm localizes sites of Ca²⁺ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.