Ratiometric calcium concentration estimation using LED excitation during mechanotransduction in single sensory neurons

In a previous study using Oregon Green BAPTA-1 fluorescence we found that intracellular calcium concentration in spider mechanoreceptor neurons rose during mechanical stimulation. We also showed that calcium elevation required the opening of voltage-dependent calcium channels by action potentials, and could not be produced by the receptor potential alone. While evidence for mechanisms of calcium elevation in these neurons was clear, our estimates of actual calcium concentration depended on properties of the fluorescent dye in the neuron cytoplasm that could not be verified. We have now developed a method for ratiometric estimation of calcium concentration in these neurons using Fura Red dye, excitation by two light emitting diodes (LEDs) of different wavelengths, and an avalanche photodiode fluorescence detector. The method is simple and economical to implement, allows concentration changes to be measured in the millisecond time range, and could easily be applied to a wide range of preparations. Resting calcium concentration in these neurons was about 70 nM and rose to a maximum of about 400 nM at firing rates above 20 action potentials per second.