Anellated hemicyanine dyes in a neuron membrane: Molecular Stark effect and optical voltage recording

The voltage sensitivity of hemicyanine dyes ANNINE-6 and ANNINE-5 with anellated benzene rings and without free CC single and double bonds is studied in Retzius neurons from Hirudo medicinalis. For comparison, biaryl hemicyanine BNBIQ and styryl hemicyanines di-4-ANEPBS and RH-421 are investigated. Fluorescence spectra are recorded by an independent variation of the wavelengths of excitation and emission at two defined membrane voltages. With extracellular staining, a positive change in the intracellular voltage shifts all excitation spectra to the blue. That modulation is assigned to a molecular Stark effect that increases in the series RH-421, di-4-ANEPBS, BNBIQ, ANNINE-5, and ANNINE-6 with displacements of elementary charge by 0.24, 0.43, 0.51, 0.65, and 0.81 nm across the membrane. For BNBIQ, ANNINE-5, and ANNINE-6, an almost identical blue shift is observed for the emission that is also assigned to a Stark effect. The ANNINE dyes are the most efficient fluorescent probes of neuronal activity on the basis of a well-defined physical mechanism. The implications with respect to the optical recording of voltage transients are considered.