Cal-520FF is the Present Optimal Ca²⁺ Indicator for Ultrafast Ca²⁺ Imaging and Optical Measurement of Ca²⁺ Currents

Ultrafast Ca²⁺ imaging using low-affinity fluorescent indicators allows the precise measurement of the kinetics of fast Ca²⁺ currents mediated by voltage-gated Ca²⁺ channels. Thus far, only a few indicators provided fluorescence transients with sufficient signal-to-noise ratio necessary to achieve this measurement, with Oregon Green BAPTA-5N exhibiting the best performance. Here we evaluated the performance of the low-affinity Ca²⁺ indicator Cal-520FF to record fast Ca²⁺ signals and to measure the kinetics of Ca²⁺ currents. Compared to Oregon Green BAPTA-5N and to Fluo4FF, Cal-520FF offers a superior signal-to-noise-ratio providing the optimal characteristics for this important type of biophysical measurement. This ability is the result of a relatively high fluorescence at zero Ca²⁺, necessary to detect enough photons at short exposure windows, and a high dynamic range leading to large fluorescence transients associated with short Ca²⁺ influx periods. We conclude that Cal-520FF is at present the optimal commercial low-affinity Ca²⁺ indicator for ultrafast Ca²⁺ imaging applications.