Fig. 5

Comparison of different fluorescent dyes in the labeling reaction. A Sketch of labeling with different fluorescent dyes, emitting in wavelength bands represented by the used colors. Created with BioRender.com. B Labeling efficiency of Atto 565 and Abberior STAR 635p at two different couples of dyes-Sfp concentrations (reaction time: 20 min). C Efficiency for Atto 565 compared with Atto 488, Alexa 488, and Abberior STAR 488 for 20-min reactions in fixed reaction conditions. ***P < 0.001, **P < 0.01, 1-way ANOVA, Bonferroni multiple comparisons. D Experiments for measurement of Atto 488 efficiency in variable conditions. The first labeling is performed with the test probe Atto 488, the second labeling is performed with the control probe Atto 565, the latter in conditions of known efficiency. Each image column shows a single cell: DIC image (top), TIRF image of Atto 488 channel (middle, green), TIRF image of Atto 565 channel (bottom, orange). Eff.: estimated efficiency. Scale bar: 5 μm. E Efficiency comparison between Atto 488 and Atto 565. Left: comparison at different dye concentrations ([Sfp]: 10 µM, time: 20 min). ***P < 0.001, **P < 0.01, Welch test. Right: comparison at different reaction times at the indicated dyes concentrations ([Sfp]: 10 µM). F Comparison of non-specifically adsorbed spot density for Atto 488 and Atto 565, using the same conditions as in E. Results on Atto 565, already shown in previous Figs. 2, 3, and 4, are here reported for direct comparison. ***P < 0.001, **P < 0.01, Welch test. In B, C, E, and F, data are mean ± SEM and are obtained from 13 to 30 analyzed cells from two independent replicates