, 1997), unless of course one injects the dye intracellularly (see below). In cultured mammalian preparations, see more however, voltage imaging of populations of neurons with single-cell resolution is possible after bath application of the fluorophores (Grinvald and Farber, 1981). In terms of the use of organic voltage-sensitive dyes for probing subcellular compartments, one can microinject the fluorophores into isolated cells in brain

slices, and after a relatively long wait for diffusion to occur, necessary for the fluorophore to distribute along the inner leaflet of the plasma membrane of the neuron, one can image dendritic voltage responses with enough signal to noise to visualize action potentials in dendrites and in spines with one-photon- and two-photon-induced fluorescence (Figures selleck screening library 3B and 3D; Antic and Zecevic, 1995 and Holthoff et al., 2010). The high lipophilicity of these fluorophores makes experiments difficult, because if any chromophore is released accidentally near the site of interest, it binds indiscriminately to all surrounding membranes, resulting in a strong fluorescent

background, which contaminates the signal of interest. The lipophilic nature can be advantageous, however, as once inside the membrane the fluorophores migrate along the membrane and can be exploited for use as tracers for anatomical pathways and to enhance the staining (Wuskell et al., 1995, Biophys. J., abstract). Finally, there has been an effort to synthesize newer families of red-shifted probes with good voltage sensitivity that are well suited for both one- and two-photon excitation (Kuhn et al., 2004), therefore enabling the

high-resolution voltage measurements from highly scattering media, with the optical sectioning capabilities afforded by nonlinear excitation. Fluorescent proteins, most of them variants of the green fluorescent protein Amine dehydrogenase (GFP), have become widely used for in vivo cell labeling (Chalfie et al., 1994 and Tsien, 1998). Combined with protein moieties that provide specific binding to a ligand, they can be engineered to report changes in intracellular free calcium and in other ions or small metabolites (Miyawaki et al., 1997 and Tsien, 2009). Because they are genetically encoded, these probes enable the genetic labeling and specific targeting of the chromophore, properties that are ideal for their use in vivo. There have been several different attempts to build voltage-sensitive fluorescent proteins. Most use a voltage-sensitive domain of an ion channel, or of another protein, as the voltage sensor that sits in the plasma membrane and experiences the electric field.