P.W. TINNING∗, A.J.P.M. FRANSSEN‡, S.U. HRIDI‡, T.J. BUSHELL‡& G. MCCONNELL†
Biophotonics, Cell Biology, Electrophysiology, Neuroscience, Optogenetics
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. TheLEDs 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 ofFura-2. Using this illuminator, we have shown that Fura-2acetoxymethyl ester (AM) concentrations as low as 250 nMcanbeusedtodetectinducedCa2+eventsintsA-201cellsandwhile utilising the 150μs switching speeds available, it waspossible to image spontaneous Ca2+transients in hippocam-pal neurons at a rate of 24.39 Hz that were blunted or absentat typical 0.5 Hz acquisition rates. Overall, the sensitivity andacquisition speeds available using this LED illuminator signif-icantly improves the temporal resolution that can be obtainedin comparison to current systems and supports optical imag-ing of fast Ca2+events using Fura-2.
The peak output spectra for the 340 nm (pE-100-340,CoolLED, Andover, UK) and 380 nm LEDs (pE-100-380,CoolLED) were measured using a spectrometer (USB2000+UV-VIS-ES, OceanOptics, Florida, USA
Product Associated Features
The 340nm and 380nm LED illumination system provides the optimum excitation wavelengths for Fura-2-based calcium imaging allowing high-precision, stable, high-throughput imaging with video-rate time resolution.
Live Cell Issues
Calcium Ration imaging
Journal of Microscopy
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