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 utilising 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.
The ability to study calcium dynamics using fluorescence microscopy allows a large population of cells to be studied simultaneously and with minimum damage to the cells. One of the key fluorescent dyes that reports the quantity of calcium inside a cell, Fura‐2, has an excitation wavelength of 380 nm when unbound to calcium and 340 nm when bound to calcium. When a ratio of the fluorescence emission signal is taken at these two wavelengths, quantitative information about the intracellular calcium concentration can be obtained. Until now, fluorescence excitation of Fura‐2 had to be performed using either an arc lamp, which has poor amplitude stability and slow wavelength switching speeds, which means that fast changes in calcium may be missed, or light‐emitting diode (LED) combinations of 350/380 or 360/380 nm. Although LEDs have faster switching speeds and higher amplitude stability than arc lamps, the 350 and 360 nm wavelengths are not well matched to the narrow 340 nm excitation peak wavelength of Fura‐2. In this report, we demonstrate the first application of a 340/380 nm LED illuminator for ratiometric Fura‐2 calcium imaging of live cells. This illuminator uses wavelengths more accurately matching the peak wavelengths of Fura‐2, and we found that this conferred several advantages over existing illuminators. We found that it is possible to detect changes in intracellular calcium with a precision of better than 5 nM. Because Fura‐2 has a theoretical precision of 5–10 nM, our instrument performance was only limited by the response of the dye. We also explored the prospect of using lower concentrations of Fura‐2 than typically used in calcium imaging studies, and we observed that it was possible to use 25% of the normal dye concentration without compromising the measurement. Finally, we demonstrated the utility of rapid switching (150 s) between the 340 and 380 nm LEDs to image and quantify the change in calcium concentration during drug‐mediated calcium events in hippocampal neurons at video rate, with no obvious photobleaching or cell damage.
Keywords: Fluorescence microscopy; light-emitting diodes; medical and biological imaging; microscopy; physiology.
© 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.