Although currently used voltage imaging methods have some shortcomings, they are useful, and researchers have succeeded in measuring membrane potential in a variety of mammalian preparations. In addition, novel imaging modalities have been recently developed and, although they have not yet been implemented for voltage imaging, they could hold great promise for future work.
One example is the use of nanoparticles, such as nanocrystals or quantum dots (Hallock et al., 2005
). These are small inorganic (metal or semiconductor) particles with well-defined electronic structure and precise quantum states. Composed of many atoms or molecules, the nanoparticles can have very strong interactions with the light field, leading to very large extinction coefficients and highly efficient emission (). The specialized structure of nanoparticles enables the generation of excitons, which can be sensitive the external electric field, resulting in strong modulations in the quantum yield, spectra, or lifetime with voltage changes. Most of these particles are coated with a passivation layer or specialized shell that limits direct interaction with the surrounding media, greatly minimizing bleaching, and in the cell, the generation of reactive oxygen species. Nanoparticles could be used alone, or combined with a conventional chromophore, as under certain conditions, they have been shown to greatly enhance optical signals, acting as an “antenna” for the light (Stiles et al., 2008
; Tam et al., 2007
). Thus when coupled to nearby chromophores, there could be large increases in fluorescence, Raman, or SHG. Already, membrane-bound, antibody-linked gold nanoparticles have been used to increase SHG from single dye molecules allowing site specific measurements of membrane potential (Peleg et al., 1999
). On the negative side, nanoparticles can be large (>10 nm)) and difficult to properly deliver in biological samples, with coating procedures and functionalization seemingly more art than science. Nevertheless, if they could be properly targeted to the membrane, their optical properties and voltage sensitivity could make them ideal voltage sensors and some examples of their potential use have been published (; (Fan and Forsythe, 2008
As another potential strategy, one might be able to use other non-linear imaging modalities to optically interrogate intrinsic chromophores present in the membrane. For example, Raman imaging (Evans and Xie, 2008
), sum-frequency or third-harmonic generation (SFG, THG; (Flörsheimer et al., 1999
; Yelin and Silberberg, 1999
)) or the recently developed stimulated radiation imaging methods (Freudiger et al., 2008
; Geiger, 2009
; Min et al., 2009
), could potentially to be used to directly monitor the small spectral changes caused 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 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 trans-membrane 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 non-linear 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 non-traditional 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 it often occurs in science and particularly in biological imaging (as illustrated by the development 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. This would be an situation analogous to the use of calcium indicators, with different affinity dyes being used to measure calcium signals with different amplitudes and kinetics (Neher, 1998
; Tank et al., 1995
A final note relates to the importance of identifying cell types in this type of optical experiments. Since most mammalian circuits are composed of different cellular elements, mixed together, and since it is likely that different subtypes of neurons serve different circuit functions, it appears essential, not only to monitor voltage responses with single cell resolution, but also to distinguish the specific cell type of each imaged neurons. In this respect, the use of genetically engineered animals where subsets of cells can be specifically labeled, or targeted, seems crucial. While ideally a genetic voltage indicator could be targeted specifically to a subset of neurons, one could also perform voltage measurements using a non-genetic method in animals where cell types are previously labeled with a genetic, or non-genetic, marker.
This is an exciting moment. Reliable, quantitative voltage imaging is arguably still the biggest current technical hurdle in mammalian neuroscience and we are now, as a research field, almost there. We ourselves remain agnostic as to which of the many different approaches discussed (organic fluorophores, SHG chromophores, genetic indicators, hybrid approaches, nanoparticles, intrinsic) is the most promising one but are hopeful for all of them. Our opinion is that, rather than a “winning horse”, it seems that at this point, the race has just started and none of these techniques has a significant advantage over the others, so parallel efforts should be undertaken to improve voltage imaging, rather than focusing on a single approach. A practical goal for voltage imaging would be to measure voltage signals at the soma, for example, with a S/N of 2 for individual action potentials, without averaging, allowing detailed monitoring of spontaneous and evoked activity in a population of neurons with single-cell specificity. Similarly, the voltage associated with quantal events in individual spines should be measured with the same S/N and without averaging. These are attainable goals, and ongoing improvements in voltage sensors could quickly break the logjam and enable what could be a new era for the study of neuronal integration and mammalian circuits. All hands aboard!