J. Membrane Biol. 58, 123-137 (1981) The Journal of Membrane Biology Improvements in Optical Methods for Measuring Rapid Changes in Membrane Potential R.K. Gupta, B.M. Salzberg, A. Grinvald*, L.B. Cohen, K. Kamino**, S. Lesher, M.B. Boyle, A.S. Waggoner, and C.H. Wang*** Department of Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, Department of Physiology and Pharmacology, University of Pennsylvania, School of Dental Medicine, Philadelphia, Pennsylvania 19104, Department of Chemistry, Amherst College, Amherst, Massachusetts 01002, and Marine Biological Laboratory, Woods Hole, Massachusetts, 02543 Summary. In an effort to increase the utility of optical methods for measuring membrane potential in excit- able cells, an additional 369 dyes were tested on giant axons from the squid. Several promising dyes with relatively large absorption and fluorescence signals are described. In addition, a simple modification of the apparatus led to a sixfold increase in the size of dye-related birefringence signals. In preparations with a suitable geometry, these signals are as large as absorption signals but photodynamic damage and bleaching are eliminated when wavelengths longer than the absorption band are used. Dye-related absorption and fluorescence signals have provided a new and sometimes powerful method for monitoring membrane potential in a wide variety of preparations (for reviews see Cohen & Salzberg, 1978 ; Waggoner, 1979). Utilizing giant axons from squid as a screening preparation, we had already tested sev- eral hundred dyes looking for useful signals (Cohen et al., 1974; Ross et al., 1977). However, the applica- tion of optical techniques to neurons of ordinary di- mensions remained limited by several interrelated fac- tors: the small size of the signals, pharmacologic ef- fects, photodynamic damage, and dye bleaching. For example, the signal-to-noise ratios were small in ex- periments on invertebrate neurons in intact ganglia or on tissue cultured mammalian neurons; only large action potentials (> 50 mV) in cell bodies are easily detected in single trials (Salzberg, et al., 1977; Woo- * Present address : Department of Neurobiology, Weizmann In- stitute of Science, Rehovot, Israel. ** Present address." Department of Physiology, Tokyo Medical and Dental University School of Medicine, Bunkyo-ku, Tokyo 113, Japan. *** Present address." Abbott Laboratories, North Chicago, Illinois. lure & Strumwasser, 1978; Ross & Reichert, 1979; Grinvald etal., 1981a; Grinvald, Ross & Farber, 198lb). Measurements of synaptic potentials or mea- surement of spikes from smaller membrane areas (e.g., growth cones) usually required signal-averaging techniques. Care was needed to avoid pharmacologic effects in experiments monitoring activity that de- pended on several synaptic relays (Grinvald et al., 1981). Finally, using higher intensity illumination, or longer sweeps increases the relative rate of dye bleach- ing in tissue cultured neurons to such a degree that each record had to be corrected for this effect (Grin- vald et al., 1981a). We have continued to synthesize additional dyes and attempted to improve the measur- ing techniques in order to obviate the above prob- lems; our progress is reported here. Roman numerals are used to refer to dyes in Ta- ble 2 of this paper, Table 2 of Ross et al. (1977), and Tables 3 and 4 of Cohen et al. (1974). Arabic numer- als refer to dyes listed in the Appendices of the three papers. Preliminary reports have been published (Co- hen et al., 1977; Grinvald et al., 1978; Salzberg, 1978, 1979). Materials and Methods Giant axons with diameters of 380 to 660 gm were dissected from the hindmost stellar nelves of the squid, Loligo pealii, and cleaned of small fibers. The apparatus used for measurements of absorp- tion, birefringence, and fluorescence is shown schematically in Fig. 1; it was similar in most respects to that described previously (Ross et al., 1977). Light from a quartz-halogen tungsten-filament lamp was passed through heat and interference filters (filter 1) and focussed to a 5-ram spot centered on the axon. Barrier filters (filter 2) passed the emitted fluorescence and prevented scattered incident light from reaching the photodetector in the fluorescence measurement. For the absorption and birefringencemeasurements, an image of the axon was formed with a microscope objective and a variable slit was positioned in the image plane parallel to the axis of the axon so that only light passing through the axon reached the detector. For birefringence experimentsa calcite Glan- 0022-2631/81/0058-0123 $03.00 9 1981 Springer-Verlag New York Inc.