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

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 ms) 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.
Source: Journal of Microscopy - Category: Laboratory Medicine Authors: Tags: Original Article Source Type: research