Fluorescence Fluctuation Microscopy Protocol

FCCS was performed on a Zeiss LSM 880 Airyscan confocal microscope equipped with a Zeiss C-Apochromat 40x, numerical aperture 1.2, water immersion objective. Image acquisition and measurement point selection were controlled by Zen Black software. Excitation was provided by the 488 nm laser line of an Argon ion laser and 561 nm laser line of HeNe laser. The laser power, measured before the objective, was 3 μW for 488 nm and 9 μW for 561 nm. This unequal power was selected to reduce the relative magnitude of green fluorescence bleed-through into the red channel (Jülich et al., 2015 (link)). The emitted light passed through a 34 μm pinhole and was separated by MBS 488/561/633 into two different detection ranges of 508–535 nm for the green channel and 606–668 nm for red channel set for internal 32-Channel GaAsP array. The correlator was set as 0.2 ms binning with 8 tau channels. The acquisition time for a measurement was 10 s.