Even in ideal situations, the optical detection of the membrane potential can only be carried out with relatively few emitted photons. Because of this, for the signal to be distinct from the photon shot noise, one typically needs to use very efficient chromophores, very strong light sources, or extensive Dabrafenib mw temporal or spatial averaging. Unfortunately, despite its great strength as an insulating layer and in maintaining cellular integrity, the plasma membrane is also a very delicate part of the cell and does not tolerate intense illumination. The photodamage associated with excited state reactions,

such as the generation of disruptive oxygen free radicals and other triplet state reactions, or simply by local heating, following photoabsorption by the chromophores used to measure the voltage signals, can easily

compromise the integrity of the membrane and kill the cell. Indeed, some sort of photodamage is present in essentially all voltage imaging measurements and is find protocol normally the reason voltage imaging experiments are terminated. To make this situation worse, neurons, like most mammalian cells, have a significant complement of endogenous chromophores, such as flavins, cryptochromes, and phorphyrins, that absorb visible light and, in some cases, are even located near the membrane. So even illuminating unstained neurons can lead to the generation of oxygen free radicals, damaging the membrane and altering membrane conductances, and may even result in membrane perforations ( Hirase et al., 2002). This endogenous photodamage is so prevalent that one sometimes wonders whether neurons have light-sensing machinery, as unicellular organisms do, to monitor circadian light changes. A third constraint arises from the fact that most of the membranes in cells are actually internal membranes. The plasma membrane, the only one across which the neuronal

membrane potential exists, is only a small proportion of the total membrane surface in the neuron. Thus, any chromophore that binds indiscriminately to membranes will mostly bind to internal membranes, which have no direct sensitivity to the plasma membrane voltage, and as a result, these Sitaxentan chromophores will merely contribute to the background noise of the measurement. This is quite a significant problem, one that again does not exist for calcium imaging, where the intracellular calcium eventually equilibrates by diffusion in the cytoplasm, in principle making every molecule of chromophore in the cytosol a possible contributor to measuring the signal. For voltage imaging, the desire to target only the plasma membrane and yet avoid internal membranes compounds the already strong localization requirements.