Measurement of intracellular ion dynamics with microfluorescent ratio imaging system

To study the ionic dynamics of spatial distribution and cellular signaling, we have setup a microfluorescent ratio imaging system for quantitative measurement of intracellular calcium and proton concentration with fluorescent indicator, Fura-2 and BCECF. Combined with cell culture techniques, this setup can measure responses from single cell and cell to cell interactions in time lapse mode. The in-homogeneous distribution of intracellular calcium ions under resting condition and after chemical stimulus can also be clearly seen and subjected to quantitative measurement. The results indicate that the resting level of calcium is around 90 nM before maturation (2 DIV.) while significantly raised to around 110 nM after maturation (7 DIV.) in cultured cerebellar granule neurons. Thapsigargin (Thsg) does not transient increase intracellular calcium level as reported in other cell preparations.