Determination of labeling efficiency, performed as described in Fig. 1 A, for Abberior STAR 635p (Abb.635, red channel) in conditions c B , while using two different conditions (c A on the left and c A on the right) for reactions with the other probe (Atto 565, orange channel). Each image column shows a single representative cell: DIC image (top) and TIRF images of the channel of the dye used in the first (middle) and second (bottom) labeling reaction. Conditions for Atto 565 were as follows: 100 nM CoA-dye, 2 µM Sfp, and 20 min reaction time (left) and 100 nM CoA-dye, 10 µM Sfp, and 10 min (right); conditions for Abb.635 were in both cases 40 nM CoA-dye, 10 µM Sfp, 20 min. r and r’ are the ratios between the number of molecules labeled in the two channels. Reported efficiency results are mean ± SEM estimate (see the “ Methods ” section), obtained from 17, 20, 25, and 16 cells analyzed for experiments represented in each image column. The results obtained for Abb.635 efficiency are not significantly different, and the efficiency estimated averaging the two results is 32.7 ± 1.7%.
