title: An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging.
abstract: Local Ca(2+) transients such as puffs and sparks form the building blocks of cellular Ca(2+) signaling in numerous cell types. They have traditionally been studied by linescan confocal microscopy, but advances in TIRF microscopy together with improved electron-multiplied CCD (EMCCD) cameras now enable rapid (>500 frames s(-1)) imaging of subcellular Ca(2+) signals with high spatial resolution in two dimensions. This approach yields vastly more information (ca. 1 Gb min(-1)) than linescan imaging, rendering visual identification and analysis of local events imaged both laborious and subject to user bias. Here we describe a routine to rapidly automate identification and analysis of local Ca(2+) events. This features an intuitive graphical user-interfaces and runs under Matlab and the open-source Python software. The underlying algorithm features spatial and temporal noise filtering to reliably detect even small events in the presence of noisy and fluctuating baselines; localizes sites of Ca(2+) release with sub-pixel resolution; facilitates user review and editing of data; and outputs time-sequences of fluorescence ratio signals for identified event sites along with Excel-compatible tables listing amplitudes and kinetics of events.
authors.affiliation: Department of Neurobiology & Behavior, University of California, Irvine, CA 92697, USA.
authors.forename: Kyle L
authors.initials: KL
authors.lastname: Ellefsen
authors.affiliation: Department of Neurobiology & Behavior, University of California, Irvine, CA 92697, USA.
authors.forename: Brett
authors.initials: B
authors.lastname: Settle
authors.affiliation: Department of Neurobiology & Behavior, University of California, Irvine, CA 92697, USA; Department of Physiology & Biophysics, University of California, Irvine, CA 92697, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Parker
authors.affiliation: Department of Neurobiology & Behavior, University of California, Irvine, CA 92697, USA. Electronic address: ismith@uci.edu.
authors.forename: Ian F
authors.initials: IF
authors.lastname: Smith
mesh_terms: D000465:Algorithms; D001331:Automation; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D005453:Fluorescence; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
chemical_list: D002118:Calcium
keywords: Algorithm; Automation; Calcium; Fluorescence; Imaging; Total internal reflection microscopy