LED illumination system launched for ratiometric calcium imaging

Peter Tinning of The University of Strathclyde has been working in partnership with CoolLED to develop LED technology in fluorescence and will be presenting his poster 'A 340/380 nm light emitting diode illuminator for Fura-2 AM ratiometric calcium imaging of live cells with better than 5nM precision' at MMC in Manchester this year.

Intracellular calcium plays an important role in many biological processes including the release of neurotransmitters in the brain, triggering insulin release in the pancreas, and the regulation of muscle contractions. Measuring the response of calcium concentration in response to external stimuli is critical in determining the role of calcium in biological processes. Use of ratiometric calcium indicators, such as Fura-2, allows quantification of intracellular calcium concentration but relies on efficient excitation or detection at two separate wavelengths. This work describes the use of a bespoke 340/380nm LED illumination system for Fura2 calcium imaging.

Until recently, the response time of illumination systems used for Fura-2 imaging have been limited to milliseconds due to mechanical switching of the wavelengths in arc lamp and monochromator systems. Utilizing the intrinsic µs switching speeds of LED technology, calcium responses have been recorded at 24.4Hz, limited by the camera used, revealing individual synaptically-driven calcium events in hippocampal neurons unresolvable by conventional monochromator measurement techniques.

In monochromator based systems, arc lamp drift instability presents a significant level of experimental noise, reducing the accuracy of any measurement. Using the 340/380nm LED-based system, which precisely matches the excitation wavelengths of the Fura-2 dye, the minimum experimental precision was found to be below that of the theoretical precision of the Fura-2 dye. As a result, the measurements were fundamentally limited by the precision of the dye rather than the optical system.

Using the LED illumination system, four times less Fura-2 dye can be loaded into the cells whilst still maintaining the same measured calcium concentration and good signal-to-noise ratio. The four-fold reduction in required dye not only improves cell-viability due to reduced dye toxicity, but also results in a large cost reduction per experiment.

Combining the above factors, the new 340/380nm LED illuminator provides the optimum excitation wavelengths for Fura-2-based calcium imaging allowing high-precision, stable, high throughput imaging with video rate time resolution.