Fig. 6

Conditions for simultaneous two-color labeling. A Sketch of the experiment for efficiency measurement in simultaneous two-color labeling. The test probe is a mix of Atto 565 and Atto 488; the control probe is Abberior STAR 635p. Created with BioRender.com. B Experimental TIRF images acquired after the two labeling reactions. For each of the two representative shown cells, we reported at the top the Atto 488 (green) and Atto 565 (orange) channels, with estimated total efficiency for the two-color labeling; at the bottom the Abberior STAR 635p channel (red); scale bar: 5 μm. C Results in different reaction conditions as described above the graphs for each column; top: measurement of two-color labeling efficiency (mean ± SEM), bottom: ratio between the number of molecules labeled with Atto 488 and with Atto 565 (box: mean ± SEM, whiskers: standard deviation, dots: averages, diamonds: individual data from two independent repetitions). ***P < 0.001, **P < 0.01, 1-way ANOVA, Bonferroni multiple comparisons. ^###P < 0.001, Welch test. D Example of simultaneous two-color single-molecule imaging and single-particle tracking analysis at high labeling efficiency in the optimized conditions. Left: first TIRF image of the acquired movie where the same cell is simultaneously detected in the Atto 488 channel (left) and the Atto 565 channel (right). Right: superimposed yellow tracks as obtained with single-particle tracking after splitting and analyzing each channel (shown is the 50th frame with tracks reconstructed until that frame from a 100-frame movie; see also Additional file 4: Video S3, Additional file 5: Video S4, Additional file 6: Video S5). Scale bars: 5 μm