title: Imaging local Ca2+ signals in cultured mammalian cells.
abstract: 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.
authors: Neurobiology and Behavior, University of California, Irvine.
authors: Jeffrey T
authors: JT
authors: Lock
authors: Neurobiology and Behavior, University of California, Irvine.
authors: Kyle L
authors: KL
authors: Ellefsen
authors: Neurobiology and Behavior, University of California, Irvine.
authors: Bret
authors: B
authors: Settle
authors: Neurobiology and Behavior, University of California, Irvine; Physiology and Biophysics, University of California, Irvine.
authors: Ian
authors: I
authors: Parker
authors: Neurobiology and Behavior, University of California, Irvine; ismith@uci.edu.
authors: Ian F
authors: IF
authors: Smith
mesh_terms: D000465:Algorithms; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling; D002460:Cell Line; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D012984:Software
chemical_list: D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium