Figure 4.

Fluorescence-based methods to characterize viscosity and surface tension of condensates. (a) FRAP is performed by bleaching a region (disk, strip, whole) of the condensate. The FRAP recovery curves are typically fitted by a single expoenential function which returns a single characteristic time scale of protein rearrangement within the droplet. This time scale can be translated into an apparent diffusion coefficient of the proteins D = R²/τ (see ref (39)) which in turn may be used to crudely estimate the apparent viscosity η via the Stokes–Einstein relation.⁵ (b) FCS is performed by detecting fluorescent molecules (typically GFP or proteins) diffusing through a small (∼femtoliter) volume within the sample. After fitting the autocorrelation function G(t) the apparent diffusion constant is obtained as D = w_xy²/(4τ), where w is the size of the sampled volume. A big advantage of FCS over FRAP is that it can quantitatively measure the concentration of molecules in the illuminated volume. (c) Passive droplet coalescence can quantify the relaxation time scale of droplets. Assuming pure Newtonian behavior, the balance of viscosity and surface tension determines the typical relaxation time scale as τ = η/γ L. Active droplet coalescence, done by pushing together droplets trapped by optical tweezers, can overcome some technical problems of passive coalescence experiments.