Lsm 780 confocor 3

FCS measurements were performed with a microscope (LSM 780 ConfoCor 3; Carl Zeiss). GFP and Alexa Fluor 488 fluorescent proteins (FPs) were excited with 488 nm from the built-in Ar ion laser. An acousto-optical tunable filter was used to adjust the incident irradiance after the microscope objective (40× C-Apochromat, NA 1.2, water immersion; Carl Zeiss) below 2 kW/cm² for the Ar ion laser (∼0.4% laser power) to reduce photobleaching and photophysical effects. Systematic determination of the lateral beam waist (ω₀) of the focused laser and the structural parameter S was performed by measuring the translational diffusion time constants τ_D of 50 nM of soluble Alexa Fluor 488 (Life Technologies) with known diffusion coefficients D in aqueous solution (435 µm²/s) according to ω₀ = (4D × τ_D)^1/2. We obtained ω₀ = 204 nm and S = 5.5, yielding effective volumes of 0.3 fl. The values of S and ω₀ were kept constant for the fitting of the different samples. Thereafter, fluorescence intensity time traces and correlation curves of iU2OS cells, expressing either the different constructs, were recorded for 45 s. Cells with too high expression levels or presenting substantial fluorescence heterogeneity were not considered further.

All FCS measurements were performed at 20°C on a point-scanning confocal microscope (Zeiss LSM780 Confocor3) equipped with a UV-VIS-IR C Achromat 40× 1.2 NA water-immersion objective and a gallium arsenide detector array wavelength selected between 491 and 561 nm. Before each experiment, the system was aligned using a high concentration and calibrated using a low concentration of rhodamin 6G solution in water. The known diffusion coefficient of rhodamine 6G (410 µm²/s; Majer and Zick, 2015 (link)) allowed us to determine the lateral beam waist (w_xy = 232 nm) and the structure factor (S = 5.77) of the focused laser (PSF). The resultant volume of illumination is calculated through the following formula: $\begin{array}{l}{V}_{eff}{ = pi}^{\mathrm{(3/2)}}{{· \mathrm{w}}_{\mathrm{xy}}}^2{· \mathrm{w}}_{\mathrm{z}}{= \mathrm{pi}}^{\mathrm{(3/2)}}{{· \mathrm{w}}_{\mathrm{xy}}}^2{· \mathrm{S}·\mathrm{w}}_{\mathrm{xy}}\\ = 0.401 {\mathrm{\mu m}}^3= 4.01{ · 10}^{-16} \mathrm{liter}\mathrm{.}\end{array}$ The values for w_xy and S were used as constants in the subsequent model-based fittings of the ACFs, and the volume was used to calculate the concentration (see below).