A large change in dye absorption during the action potential.

Recently, we began a search for easily detected changes in the optical properties of neurons that occur during excitation (1, 2). Until now, changes in extrinsic fluorescence were the most sensitive indicators of neuronal activity. More than 500 fluorescent dyes have been examined on giant axons from squid and a number were found (2) which gave fluorescence changes during single action potentials that were significantly larger than the noise in the measurements. Although it was subsequently demonstrated that one of these fluorescence signals was large enough to allow optical monitoring of action potentials in individual neurons of a leech segmental ganglion (3), larger signals would facilitate more complex experiments of this kind. Previous results (4-8) had suggested that there might be changes in light absorption of dyed axons during activity. We have now found absorption changes during the action potential using a number of dyes and several of these could be measured with a signal-to-noise ratio considerably larger than fluorescence changes obtained under the same conditions. Giant axons (diameters 320-500 ,m) from the squid, Loligo pealii, were cleaned of small fibers and placed horizontally in a chamber containing two pairs of platinum electrodes for stimulating and extracellular recording. The chamber was mounted on the stage of a Leitz Ortholux II microscope (E. Leitz, Inc., Rockleigh, N.J.). Light from a quartz-halogen tungsten-filament lamp was collimated, made quasimonochromatic with a bandpass interference filter, and focused on the axon by means of a bright-field condenser. Light collected by a 20 x (NA 0.4) objective was passed through a slit in the objective image plane which limited the light reaching a SGD-444 photodiode (E G & G, Inc., Salem, Mass.) to that originating from a 150 ,m length of axon. The axons were incubated with a solution of dye in seawater (2) for 20 min and then bathed in seawater bubbled with argon. In a few axons the intracellular potential was measured by impaling the axon with a glass microelectrode (filled with