Porphyrins for Probing Electrical Potential Across Lipid Bilayer Membranes by Second Harmonic Generation**

Neurons communicate by using electrical signals, mediated by transient changes in the voltage across the plasma membrane. Optical techniques for visualizing these transmembrane potentials could revolutionize the field of neurobiology by allowing the spatial profile of electrical activity to be imaged in real time with high resolution, along individual neurons or groups of neurons within their native networks. Second harmonic generation (SHG) is one of the most promising methods for imaging membrane potential, although so far this technique has only been demonstrated with a narrow range of dyes. Here we show that SHG from a porphyrin-based membrane probe gives a fast electro-optic response to an electric field which is about 5–10 times greater than that of conventional styryl dyes. Our results indicate that porphyrin dyes are promising probes for imaging membrane potential. Studies of excitable cells, such as neurons and cardiac myocytes, require new methods for mapping changes in membrane potential, ideally with a voltage resolution of about 1 mV, a time resolution of 1 ms, and a spatial resolution of 1 mm. Microelectrodes can be used to measure transmembrane potentials with excellent sensitivity and temporal resolution, but they do not provide subcellular spatial resolution. Fluorescent calcium indicators are widely used to probe membrane potential indirectly, through the intracellular Ca concentration. However, changes in calcium concentration do not accurately reflect voltage transients. Fluorescent voltage-sensitive dyes were first developed 30 years ago, and recent advances in the molecular design of these dyes have made it possible to track the spatial evolution of action potentials. Compared with fluorescence, SHG imaging has several advantages as a technique for probing membrane potential: 15–21] SHG has a greater intrinsic sensitivity to electric fields than fluorescence, and it is only produced by noncentrosymmetric molecules in noncentrosymmetric environments, thus making it ideal for probing interfaces such as lipid bilayers. Similar to twophoton-excited fluorescence, SHG is a two-photon nonlinear optical effect, and it brings the advantages of depth penetration associated with multiphoton microscopy. The main disadvantage of SHG is that it often gives lower intensity than fluorescence; there is a need for the design of brighter, more voltage-sensitive SHG dyes. Styryl dyes and retinal chromophores have been shown to exhibit voltage-sensitive SHG when localized in plasma membranes, and the voltage sensitivity of their SHG is greater than that of their fluorescence. Recently we reported that amphiphilic porphyrins such as JR1 exhibit strong SHG when localized in lipid membranes. Here we compare the voltage sensitivity of the SHG from JR1 with that from three widely studied styryl dyes (FM4-64, di-4-ANEPPS, and RH237) in hemispherical lipid bilayers (HLBs). The results show that the SHG signal from the porphyrin-based dye is exceptionally sensitive to an electric field.