High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes

An improved method for making fast quantitative determinations of membrane potential with voltage-sensitive dyes is presented. This method incorporate s a high-speed,random-access, laser-scanning scheme (Bullen et al., 1997. B iophys. J. 73;477-491) with simultaneous detection at two emission waveleng ths. The basis of this ratiometric approach is the voltage-dependent shift in the emission spectrum of the voltage-sensitive dye di-8-butyl-amino-naph thyl-ethylenepyridinium-propyl-sulfonate (di-8-ANEPPS). Optical measurement s are made at two emission wavelengths, using secondary dichroic beamsplitt ing and dual photodetectors (<570 nm and >570 nm). Calibration of the ratio metric measurements between signals at these wavelengths was achieved using simultaneous optical and patch-clamp measurements from adjacent points. Da ta demonstrating the linearity, precision, and accuracy of this technique a re presented. Records obtained with this method exhibited a voltage resolut ion of similar to 5 mV, without any need for temporal or spatial averaging. Ratiometric recordings of action potentials from isolated hippocampal neur ons are used to illustrate the usefulness of this approach. This method is unique in that it is the first to allow quantitative determination of dynam ic membrane potential changes in a manner optimized for both high spatiotem poral resolution (2 mu m and <0.5 ms) and voltage discrimination.