High affinity binding of fluorescence labeled hongotoxin (HgTX1-Cy5) to the potassium channel KV1.3 in T-lymphocyte cell membranes was utilized for imaging single ion channels optically, employing Single Dye Tracing (SDT). Binding sites were seen as single fluorescence peaks in cross-sections through the cell. Their number matched, at conditions of saturated binding, the number of sites expected from biochemical determination. By fitting the peaks to the pointspread-function, well approximated by a Gaussian distribution, resolution of channel positions to within ±40nm was obtained in all three dimensions. Within the focal plane (x-y plane) positional resolution is given by the accuracy of determining the peak position of the Gaussian. The positional resolution along the optical axis (z-direction) was obtained from the accuracy of estimating the position of minimum defocusing for a single molecule. For this, the width of the fluorescence peaks in consecutive images, taken at different degrees of defocusing, were shown to accurately match the theoretical prediction, yielding ~40nm accuracy of finding the z-position of the labeled channels. This first visualization of individual membrane proteins in live cells by fluorescence labeled ligands with 40nm 3D positional resolution opens new perspectives for the study of cellular organization and processes at the molecular level.
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