In spite of these challenges, in the last decade, EGFR inhibitor the groups of Lewis, Loew, and others have pioneered the application of SHG to living cells and to measurements

of membrane potential (Bouevitch et al., 1993, Campagnola et al., 2001, Lewis et al., 1999 and Millard et al., 2003). The strategy pursued has been the application of organic dyes, based on styryl fluorophores with distinct electrochromic properties, originally synthesized for fluorescence voltage measurements (Bouevitch et al., 1993). SHG imaging of neurons has also been performed with the membrane-trafficking dye FM4-64, enabling high-resolution measurements of voltage of somata, dendrites, and dendritic spines (Figure 4C; Dombeck et al., 2004, Dombeck et al., 2005 and Nuriya et al., 2005). As an alternative strategy to the typical chromophores, one can use trans-retinal as a SHG chromophore to measure membrane potential (Nemet et al., 2004), since it exhibits a large change in dipole

moment upon light excitation (Mathies and Stryer, 1976). Nevertheless, Dasatinib molecular weight despite advances in the rational design of chromophores for nonlinear imaging, relatively little work has gone into synthesizing chromophores specifically designed for SHG in biological samples that would maximize the SHG response while minimizing damaging alternative photoprocesses. Finally, an alternative approach to measure membrane potential relies Bay 11-7085 on intrinsic changes in the optical properties of the neurons, or axons. These approaches, which are among the earliest historically (Cohen and Keynes, 1971), are potentially very powerful because they do not need exogenous chromophores. At the same time, they can only be applied in optically very accessible preparations, such as neuronal cultures or some invertebrate preparations. Also, they generate relatively small signals and extensive averaging

is necessary. These intrinsic approaches to measure voltage have exploited different types of optical measurements, mostly in invertebrate preparations. For example, changes in light scattering, changes in optical dichroism, and changes in birefringence have been explored (Ross et al., 1977). These changes are presumably related to alteration in the refractive index or small volume changes near the membrane, in response to the rapid osmotic changes associated with ion fluxes, and have been used to monitor action potentials (Cohen and Keynes, 1971, Ross et al., 1977 and Stepnoski et al., 1991). Presumably these same intrinsic mechanisms allow for the detection of action potentials with optical coherence tomography, which uses interferometry to detect small changes in optical path length resulting from action potential activity in isolated neurons.