” As a coherent process, the SHG is strongly directed with respect to the incoming laser KU-55933 cost beam, and the signal scales

as N2, where N is the number of chromophores that are SHG active, so is strongly dependent on packing density. The strength of the overall SHG response depends on the effective χ(2), the second-order susceptibility tensor, of the system, which in turn depends on the overall alignment and microscopic properties of the chromophores. Besides its sensitivity to interfaces such as the plasma membrane, SHG appears particularly well suited for voltage imaging because, to a first-order approximation, changes in the membrane’s electric field change the effective χ(2) in a linear fashion (Table 1F), giving a direct readout of the voltage. In addition, SHG is a nonlinear parametric process and does not rely on the transfer of energy into the molecule, greatly diminishing the photodamage-associated excited state processes, such as the generation of

triplet states. Moreover, as in two-photon excitation (Denk et al., 1990), the nonlinearity of the process automatically produces optical sectioning in a laser scanning microscope, since SHG is only generated at the focal volume. This minimizes out-of-focus excitation and photodamage. Because the electro-optic mechanism of SHG is essentially Regorafenib instantaneous, the signal originates only at the membrane, and the photons are

emitted in a preferred direction with a well-defined spectral many signature, SHG seems to be the ideal method for optical recordings of membrane potential ( Jiang et al., 2007). Unfortunately, like with other voltage-sensing modalities, current implementations of SHG have been limited. Though the pure electro-optic response is fast, other slower processes that depend on voltage can affect SHG by changing the chromophores’ spectra or alignment. Over time, the chromophores responsible for SHG can equilibrate across the membrane, reducing the asymmetry of the interface and hence the overall response (Mertz, 2008). For the typical packing densities in the membrane, it is a relatively inefficient process and normally requires high peak photon fluxes (Campagnola et al., 2001 and Eisenthal, 1996), from pulsed ultrafast lasers (Millard et al., 2003). Even so, typically few SHG photos are produced, generating an overall small signal and making photon-counting measurements sometimes necessary (Jiang and Yuste, 2008). To increase the SHG signal, most experiments are done with photon energies close to an electronic resonance in the system, which enhances SHG but leads to direct photoabsorption.