Fig. 4

Labeling efficiency and nonspecific adhesion as functions of time. Conditions for single-molecule imaging at high labeling efficiency. A Labeling efficiency as a function of time measured at 1 (orange) and 10 (Bordeaux) µM of Sfp at CoA-Atto 565 concentration of 100 nM (left) and 200 nM (right). B Density of spots non-specifically adsorbed to cells and glass as a function of time measured in the same conditions of A. Dots are means, error bars are SEM estimates, and continuous lines are mono-exponential fitting curves (with obtained lifetime τ in minutes). Fifteen to 30 cells from 2 independent replicates were analyzed for each condition. C Single-molecule imaging in live cells and application of single-particle tracking analysis at found optimal labeling conditions (100 nM CoA-Atto 565, 10 µM Sfp, 10 mM MgCl₂, 20 min: efficiency 82 ± 3%). A representative cell is shown. Gray scale: TIRF images; left: first frame of the acquired 100-frame movie; right: in yellow, tracks obtained from a single-particle tracking analysis on frames 1–50, superimposed on the 50th frame (see also Additional file 2: Video S1, Additional file 3: Video S2). Scale bars: 5 μm