An interesting design for voltage-sensitive dyes is one with a mixture of organic and genetic components (Figure 2E). These hybrid strategies began with a FRET-based system, composed of an oxonol derivative that functioned as the donor and a Texas Red-labeled lectin as an acceptor (González and Tsien, 1995). Oxonols insert into the membrane and reside on one leaflet or the other depending on the membrane potential. The fluorescently labeled lectin is not membrane permeable and sits only on the outside of the membrane, and through changes in the energy transfer efficiency between the two species, it can be used GABA receptor signaling to monitor the position of the oxonol and, thus, the

membrane potential. Another strategy (Chanda et al., 2005) uses a hybrid voltage sensor (hVOS) that consists

of a molecule of GFP fused to a farnesylated and palmitoylated Lumacaftor mouse motif that attaches it to the membrane. The second component is the synthetic compound dipicrylamine (DPA) that serves as a voltage-sensing acceptor and translocates across the membrane, depending on the electric field. Unfortunately, DPA increases the membrane capacitance, so care must be taken to ensure the concentrations used do not disrupt the native physiological responses. Recently, there have been some promising results from purely chemical hybrid systems, such as the DPA-diO hybrid (Figure 4B). This combination has high sensitivity to voltage and uses low DPA concentrations, although more work needs to be done for consistent, calibrated voltage imaging in extended 4-Aminobutyrate aminotransferase experiments (Bradley et al., 2009). Hybrid strategies appear more chemically flexible than pure genetic approaches, although at the same time, they are complicated by the application of exogenous species. It can be argued that fluorescence or absorption approaches are intrinsically flawed when optically probing interfaces, because of their lack of spatial specificity

(Eisenthal, 1996). Unless a fluorophore or chromophore is selectively localized at the interface, the interface-specific signal will be greatly overwhelmed by the many other fluorophores/chromophores residing in the bulk solution, and this argument can be extended to biological membranes. SHG solves this problem by only generating signal at the interface itself (Campagnola et al., 1999, Eisenthal, 1996 and Moreaux et al., 2001). SHG is a coherent hyperscattering phenomenon by which the incoming light beam’s electric field induces a second order nonlinear polarization in the media, resulting in the emission of a photon of exactly twice the frequency (half the wavelength) of the incident photons (Figure 2F). In the asymmetric environment of interfaces, any molecules with nonsymmetric chemical or electrical properties can spontaneously align themselves with respect to the interface, whereas in solution, or the bulk media, they will be isotropically distributed and hence not oriented.