New Dyes With Fast Voltage-Dependent Changes In Membrane SHG

Abstract

Laser scanning second harmonic generation (SHG) microscopy has shown significant promise for membrane potential imaging with voltage sensitive dyes (VSDs), possessing significant advantages over fluorescence-based imaging modalities. Through simultaneous patch-clamping and non-linear imaging of cells, SHG has been found to exhibit sensitivities to trans-membrane potential that are up to four times better than those obtained under optimal conditions using one-photon fluorescence imaging (Millard et al., 2004). For styryl dyes, while electrochromism is the dominating photophysical mechanism of fluorescence, some ANEP-based dyes display slow SHG voltage responses, suggesting that chromophore membrane reorientation or redistribution may be involved. The mechanism of the SHG response is not entirely understood and necessitates additional study in order to fully optimize this imaging modality. We report on our further investigation of the time dependence of the voltage sensitivity of SHG and simultaneous two-photon fluorescence imaging, using “fast” voltage-switching experiments. The response kinetics of resonance enhanced SHG from several styryl dyes developed in our laboratory, including di-3-ANEPPDHQ, as well as di-4-ANEPPTEA and a new fluorinated derivative, have been determined. Voltage-clamped neuroblastoma cells stained with these dyes were imaged with 1064 nm excitation from a mode locked fiber-based laser source. For SHG, these VSDs were found to exhibit moderately large voltage sensitivities in addition to fast kinetic responses. Our results suggest that voltage sensitive dyes can be developed which have both large SHG signal changes and the requisite speed for use as a practical tool for measuring electrical activity in neuronal systems.(Supported by NIH grant EB001963).