FCS measurements were performed with a confocal PicoQuant (Berlin, Germany) MicroTime 200 machine and a Zeiss 880 laser scanning microscope. The microscopes provided 488 nm excitation lines focused into the sample via a 60×/1.2 (Olympus (Tokyo, Japan) UPlanSApo) and a 40×/1.2 (Zeiss C-APOCHROMAT) water immersion objective lenses, respectively. Excitation power was set to 5 μW to minimize eGFP photobleaching. Counts per molecule were maximized acting on the correction collar of the objective lenses. Time traces were recorded and FCS curves calculated for each measurement.
To measure the diffusion of eGFP and N-terminal part of PEX5L (human and from T. brucei, fused to eGFP) in solution, the proteins were diluted to ca. 10 nM. For each condition, recordings at three different spots were performed by measuring for 15–20 s five times at about 10 μm depth from the coverslip. Time traces acquired at the MicroTime were correlated using FoCuS-point software. Zeiss data were correlated using Zen software. The data were fitted using a 3D diffusion equation that includes a triplet component as described in the previous section.
To measure the diffusion of eGFP and N-terminal part of PEX5L (human and from T. brucei, fused to eGFP) in solution, the proteins were diluted to ca. 10 nM. For each condition, recordings at three different spots were performed by measuring for 15–20 s five times at about 10 μm depth from the coverslip. Time traces acquired at the MicroTime were correlated using FoCuS-point software. Zeiss data were correlated using Zen software. The data were fitted using a 3D diffusion equation that includes a triplet component as described in the previous section.