Impact of channel properties, imaging strategies and diffusional constraints on the detectability of Ca2+ microdomains

• strategies to optimize the signal-to-noise ratio in TIRF microscopy • channel properties and cell geometry define size and duration of Ca 2+ microdomains • comparative analysis of influx events through different ion channel entities • improved detection limit to visualize unitary Ca 2+ influx events Subplasmalemmal Ca 2+ microdomains formed by brief openings of single or locally clustered Ca 2+ -permeable ion channels play crucial roles in different cellular processes, including migration and secretion. While electrophysiological recordings are unmatched in sensitivity and temporal resolution, spatiotemporally resolved microfluorometric measurements of such Ca 2+ microdomains are of particular interest to identify the sites of channel activity in undisturbed cells under close-to-physiological conditions. In total internal reflection fluorescence (TIRF) microscopy, the shallow penetration depth of the evanescent field is ideally suited to monitor subplasmalemmal Ca 2+ microdomains without compromising the spatial and temporal resolution. This study systematically characterizes the impact of various imaging parameters, ion channel properties and cell geometries on the detection limits and signal-to-noise ratios in TIRF microscopy by using simulated and experimentally gathered data. The investigated ion channels include voltage-gated calcium channels, transient receptor potential channels, and P2X receptors. We provide a framework for choosing imaging parameters for high spatiotemporal resolution of Ca 2+ influx elicited by openings of single Ca 2+ -permeable cation channels of different types. Current technical limitations and potential future improvements are discussed.