For example, Raman imaging (Evans and Xie, 2008), sum-frequency or third-harmonic generation (SFG, THG; Flörsheimer et al., 1999 and Yelin and Silberberg, 1999) or the recently developed stimulated radiation imaging methods (Freudiger et al., 2008, Geiger, 2009 and Min et al., 2009) could potentially to be used to directly monitor the small spectral changes caused PD0325901 solubility dmso by the membrane potential in species intrinsic to the membrane environment, free from the constraints of exogenous labels. At the same time, these techniques would need to effectively solve the contrast problem raised above and distinguish optical signals from the plasma membrane from those of other cellular membranes.

In terms of improving

existing strategies, significant challenges need to find more be overcome. One major avenue for improvement is the rational design of novel probes, whether organic, inorganic, or genetic. For example, it is known that the exact shape of transmembrane proteins can strongly modify the local electric field, magnifying it, so that clever placement of a voltage-sensing moiety in molecular pockets where the electric field would be more concentrated could lead to an improved voltage sensor. Also, for sensors based on energy transfer, conformational changes are not the only variable affected by voltage. The rates of energy transfer also depend critically on the spectral overlap of the donor’s emission spectrum with the acceptor’s absorption spectrum, and either of these can be altered directly or indirectly as a result of changing membrane potential. Because of the highly nonlinear FRET dependence with spectral overlap of the donor-acceptor pair, it may be

more sensitive than simply monitoring the spectral changes alone. As discussed previously, current SHG based measurements suffer because of concomitant absorption and subsequent photodamage, and nontraditional chromophores with large values of χ(2) but with weak fluorescence could lead to new, useful voltage probes. It seems particularly important for research groups with extensive experience in chemistry or the physical sciences to join these efforts; as often occurs in science, and particularly in biological imaging (as illustrated by the development Terminal deoxynucleotidyl transferase of calcium indicators or of two-photon microscopy), it is from this interdisciplinary cross-fertilization that major advances are generated. In addition, more studies of the biophysical mechanisms of existing chromophores are necessary. This is not just an academic exercise, but it could be essential in the efforts to design better chromophores. Also, it should be kept in mind that there may not be a universal voltage-sensitive dye, but it could be possible to use a combination of them, depending on the kinetics of the desired signals to be measured and constraints introduced by the specific preparations.