LaminB1 labelled with Alexa 532 and microtubules labelled with Alexa 647 in Cos-7 cells using 2 different buffers (Top panel: 25mM bMEA at pH7.2; Bottom panel: 25 mM bMEA at pH5.2) displaying poor photophysics characteristics for both fluorophores (see Fig. 2b). Focus was at the top of the cell, approximately 5 μm above the coverslip surface. Acquisitions were achieved in the same conditions as for Figure 2c. As described by the photophysics parameters in Figure 2b, Alexa 647 detection was clearly affected by the decrease in thiols concentration (Top Panel) and of the pH (Bottom Panel). Also and as previously described, Alexa 532 detection is slightly affected with an apparent smaller number of localized single molecules, especially for the buffer with 25 mM bMEA, pH 5.2 (Bottom Panel).