1 January 2005 Optical single-channel recording: imaging Ca2+ flux through individual ion channels with high temporal and spatial resolution
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J. of Biomedical Optics, 10(1), 011002 (2005). doi:10.1117/1.1846074
Developments in imaging technology now enable visualization of the functioning of individual ion channels in living cells: something previously possible only by the electrophysiological patch-clamp technique. We review techniques that track channel gating via changes in intracellular [Ca2+] resulting from openings of Ca2+-permeable channels. Spatial and temporal resolution are optimized by monitoring Ca2+ close to the channel mouth, and we describe the use of two imaging modalities: confocal laser scan microscopy (linescan CLSM) and total internal reflection fluorescence microscopy (TIRFM). Both currently achieve a kinetic resolution of <10 ms, provide a simultaneous and independent readout from many channels, and enable their locations to be mapped with submicrometer resolution. TIRFM provides 2-D images from a very thin (~100 nm) optical section, but it is restricted to channels in the plasma membrane of cells adhering close to a cover glass. In contrast, CLSM can image channels in intracellular membranes but, to achieve good temporal resolution, has been utilized only in a linescan mode with limited spatial information. We anticipate that imaging techniques will develop as a useful adjunct to patch-clamping for single-channel studies, with capabilities including simultaneous readout from multiple channels, high-resolution mapping of channel location, and mobility that is inaccessible by electrophysiological means. Optical single-channel recording is applicable to diverse voltage- and ligand-gated Ca2+-permeable channels and has potential for high-throughput functional analysis.
Angelo Demuro, Ian Parker, "Optical single-channel recording: imaging Ca2+ flux through individual ion channels with high temporal and spatial resolution," Journal of Biomedical Optics 10(1), 011002 (1 January 2005). http://dx.doi.org/10.1117/1.1846074



Confocal microscopy

Channel projecting optics

Line scan image sensors

Temporal resolution

Ion channels

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