A 340/380 nm light emitting diode illuminator for Fura-2 AM ratiometric Ca²⁺ imaging of live cells with better than 5 nM precision

We report the first demonstration of a fast wavelength-switchable 340/380 nm light emitting diode (LED) illuminator for Fura-2 ratiometric Ca²⁺ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura-2 and enables the precise detection of cytosolic Ca²⁺ concentrations, which is only limited by the Ca²⁺ response of Fura-2. Using this illuminator, we have shown that Fura-2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca²⁺ events in tsA-201 cells and while utilizing the 150 µs switching speeds available, it was possible to image spontaneous Ca²⁺ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca²⁺ events using Fura-2.