As calcium is the most important signaling molecule in neurons and secretory cells, amongst many other cell types, it follows that an understanding of calcium channels and their regulation of exocytosis is of vital importance. vesicles. With combinations of these techniques the activity of individual channels can be visualized and quantified using novel Ca2+ indicators. Fluorescently labeled specific channel toxins can also be used to localize endogenous assembled channel tetramers. Fluorescence lifetime imaging microscopy and other single-photon-resolution spectroscopic approaches offer the possibility to quantify proteinCprotein interactions between populations of channels and the SNARE protein machinery for the first time. Together with simultaneous electrophysiology, this battery of quantitative imaging techniques has the potential to provide unprecedented detail describing the locations, dynamic behaviors, interactions, and Ganetespib inhibition conductance activities of many thousands of channel molecules and vesicles in living cells. oocytesSingle channel calcium fluorescence transients (SCCaFTs)TIFRM using fluorescent calcium indicator (Fluo-4)Heterologously expressed channels(29)P/Q typendndndNdnd Open in a separate window biochemistry, whilst invaluable, cannot deliver the wheres and whens of interactions in cells and so overlooks the key elements of spatio-temporal regulation. Electrophysiology can resolve single ion channel activities, or entire cell cohorts of activity, but with limited spatial resolution. It thus remains unknown how: (i) membrane cohorts of single ion channels are spatially distributed, (ii) the proportion of ion channels compared to the total pool (Ca2+ and K+) reside within functionally meaningful distances of fusion competent- and/or incompetent-vesicles, (iii) how the dynamics of channel activity may correlate with their Rabbit Polyclonal to K0100 spatial pattern interactions with the SNARE molecular machinery in intact cells, and (iv) whether every channel at the membrane is functional. It is clear that new tools are required to address these questions. Super-Resolution Imaging and Exocytosis The membrane-trafficking field has a strong history of using cutting-edge techniques and imaging is no exception. Super-resolution microscopy is an emerging powerful tool to further research on ion channels and calcium signaling involved in exocytosis, and have already been applied in studies of the exocytotic machinery (12, 38C41). Our own recent work revealed that the majority of vesicles do not access the necessary compliment of SNARE molecular machinery at the membrane required for fusion (12, 40, 42). Furthermore, vesicle dynamics are also segregated, not only spatially at the membrane but also by vesicle age as we showed that vesicles are prioritized for release according to the time since their assembly (43). This mini-review summarizes the main super-resolution imaging modalities and illustrates their potential uses in quantifying ion channel molecular biology in relation to exocytosis. Table ?Table22 summarizes the super-resolution imaging modalities described and their (potential) uses in examining ion channel biology. Table 2 Summary of available super-resolution microscopy and spectroscopic approaches and their potential for ion channel imaging. coordinates from where the signals arose; the certainty of localization is affected by brightness, noise, and pixel size. By rendering coordinates, a virtual image is created which shows the coordinate positions of all the molecules on the plasma membrane that have emitted. A variation of PALM combines single-particle tracking, so termed sptPALM (52), allowing the high-precision tracking of many 1000?s of single molecule signals with high temporal resolution. Simulated Emission Depletion Microscopy Ganetespib inhibition The underlying limitation with microscopy is the diffraction-limited width of the PSF. Single molecule localization microscopies (SMLMs) circumvent this by determining the centroids derived from single molecule PSFs, and though these are excellent techniques, they are not imaging directly sub-diffraction structures. Simulated emission depletion microscopy (STED) presents a fundamentally different approach, directly manipulating the PSF through Ganetespib inhibition the use of stimulated emission to deplete fluorophores before they fluoresce (53). On a conventional scanning microscope, the sample is raster scanned with an excitation beam, which increases the energy of the fluorophores from the ground state (70, 71) and is a specific N-type calcium channel blocker. This toxin has been used in.