Summary. The fluorescence of dyes added to squid giant axons was studied during action potentials and voltage-clamp steps. One goal was to find fluorescence changes related to the increases in membrane conductance that underlie propagation. A second goal was to find large changes in fluorescence that would allow optical monitoring of membrane potential in neurons and other cells. Attempts were made to measure fluorescence changes using over 300 different fluorescent molecules and positive results were obtained with more than half of these. No evidence was found that would relate any of the fluorescence changes to the increases in membrane conductance that accompany depolarization; most, instead, were correlated with the changes in membrane potential. The fluorescence changes of several dyes were relatively large; the largest changes during an action potential were 10 -3 of the resting intensity. They could be measured with a signal-to-noise ratio of better than 10:1 in a single sweep.Changes in the fluorescence of molecules added to axons have been studied in an effort to learn about alterations in membrane structure that occur during excitation (Tasaki, Carnay &Watanabe, 1969;Cohen, Landowne, Shrivastav &Ritchie, 1970; Conti, Tasaki &Wanke, 1971). Although it was suggested that ANS and TNS fluorescence changes were related to increases in membrane conductance, our experiments (Davila, Cohen, Salzberg &Shrivastav, 1974) indicated that these fluorescence changes were, in fact, potential dependent. With the hope that other dyes would provide information about the structural basis of the conductance
The absorption, fluorescence, dichroism, and birefringence of stained squid axons were measured during action potentials and voltage clamp steps in an effort to find large optical signals that could be used to monitor membrane potential. Changes in all four optical properties were found that were linearly related to membrane potential and, with several new dyes, the signal-to-noise ratios were larger than any obtained previously. The problem of photodynamic damage was greatly diminished; with a merocyaninerhodanine dye, the photodynamic damage associated with intense light and the presence of oxygen was negligible. The absorption change obtained with this dye was relatively large; it could be measured with a signal-to-noise ratio of 100:1 during a single action potential.
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