Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution

Imaging the quantal substructure of single IP 3 R channel activity during Ca 2+ puffs in intact mammalian cells

“…1A, gray trace), on which it was not possible to resolve the underlying step-wise changes that would be expected as individual channels stochastically open and close. To then achieve single-channel resolution, we used TIRF imaging of a subset of puff sites located close to the plasma membrane (17), so that rapid binding of Ca 2ϩ to the fast indicator dye within attoliter cytosolic volumes around puff sites yields fluorescence signals that more closely track instantaneous Ca 2ϩ flux through IP 3 R channels (14). Additionally, we gained a further improvement by loading cells with EGTA, a Ca 2ϩ buffer that inhibits wave propagation (17)(18)(19) and accelerates the collapse of the local Ca 2ϩ micro-domain but, because of its slow binding kinetics, minimally perturbs local free [Ca 2ϩ ] within a cluster (14) and has little effect on peak puff amplitudes (17)(18)(19).…”

“…Although specific instances of single IP 3 R channel fluorescence signals have been reported as isolated blips (7,13) and as ''trigger'' events immediately preceding puffs (10), it had not been possible to directly dissect the contributions of individual channels during puffs. Here, we capitalize on experimental and theoretical findings that the resolution of local Ca 2ϩ signals can be dramatically improved by monitoring fluorescence from attoliter volumes around open channels by means of total internal reflection fluorescence (TIRF) microscopy (14). We had previously used this approach to image single-channel calcium fluorescence transients arising from gating of Ca 2ϩ -permeable channels in the plasma membrane (15,16).…”

Imaging single-channel calcium microdomains

“…On the basis of modeling studies by Shuai & Parker [30], we illustrate schematically in Fig. 1 how Ca 2+ flux through a single channel leads to the generation of experimentally-observed SCCaFTs.…”

“…A number of studies have modeled Ca 2+ diffusion in the cytosol during channel opening with the aim of predicting the rapid time course of Ca 2+ -dependent processes such as transmitter release and Ca 2+ -induced receptor trafficking [28,29]. More recently, theoretical approaches have been extended to consider the relation between the free Ca 2+ microdomain around a channel and the resulting fluorescence signal, so as to aid interpretation of the fluorescence images and guide improvement of the imaging techniques [30,31].…”

Imaging the dynamics of individual electropores

“…Here, we seek to improve our experimental understanding of electroporation by exploiting optical single-channel recording (oSCR) (28,29) to image individual voltage-induced defects in a lipid membrane by detecting a fluorescent signal proportional to the flux of Ca 2+ flowing through a pore. Very recently, we detected electroporation events using fluorescence signals generated by K + ionic flux (30).…”