Action-Spectra of Electrochromic Voltage-Sensitive Dyes in an Intact Excitable Tissue

Abstract

Voltage-sensitive dyes VSDs provide a spatially resolved optical read-out of electrical signals in excitable tissues. Several com- mon fluorescent VSDs display electrochromic shifts of their emission spectra, making them suitable candidates for ratiometric measure- ments of transmembrane voltages. These advantages of VSDs are tem- pered by tissue-specific shifts to their fluorescence emission. In addi- tion, the optimal electrochromic dye response occurs in wavelength bands distinct from the dye's maximal resting emission. This spectrum can undergo tissue-specific shifts as well. We have devel- oped a technique for in situ measurements of the action spectra of VSDs in intact excitable tissues. Fluorescence emission spectra of VSDs during action-potential depolarization were obtained within a single sweep of a spectrophotometer equipped with a change-coupled device CCD array detector. To resolve the subtle electrochromic shifts in voltage-induced dye emission, fluorescence emission spectra measured right before and during field-induced action-potential de- polarization were averaged over about 100 trials. Removing white- noise contributions from the spectrometer's CCD detector/amplifier via low-pass filtering in Fourier space, the action spectra of all dyes could be readily determined. © 2008 Society of Photo-Optical Instrumentation En-