P.W. Tinning (1), A.J.P.M. Franssen (2), S.U. Hridi (2), T.J. Bushell (2) & G. McConnell (3)
(1) Department of Physics, SUPA University of Strathclyde, Glasgow, U.K.
(2) Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.
(3) Centre for Biophotonics, University of Strathclyde, Glasgow, U.K.
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. 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 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 340 nm and 380 nm 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.
Journal of Microscopy
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