Vc plan apo 60x 1.4 na oil objective

Unless otherwise stated, all proteins used in experiments contain 10:1 unlabeled to GFP-labeled versions. To lower salt concentration, proteins were mixed with no salt buffer (50 mM Tris pH8, 1mM DTT) to obtain desired protein and salt concentrations. For RNA experiments, proteins were diluted with droplet buffer (50 mM Tris pH8, 1mM DTT, 150mM KCl) to desired concentration and then unlabeled RNA or cy3-labeld RNA were added. All mixing was performed at room temperature. The mixing and imaging was done in glass wells (Grace Bio-Labs) that were treated with 30mg/ml BSA (Sigma) for 30min. Screening for droplets was done on a spinning disc confocal microscope (Nikon), with VC Plan Apo 60X/1.4 NA oil objective.

Droplet formation movies were recorded on a spinning disc confocal microscope (Nikon) with VC Plan Apo 60X/1.4 NA oil objective. Fusion events were manually spotted and cropped out in ImageJ. The cropped image series containing fusion events were analyzed in MATLAB with Imaging Processing Toolbox. As we have previously described (Brangwynne et al., 2011 ), we plot the change in aspect ratio = l/w, where l is the long axis and w is the short axis of fusing droplets, over time. The decay of aspect ratio over time was fitted to an exponential curve to obtain decay time t (Fig. S4). Plotting the decay time against droplet characteristic length
$\ell =\sqrt{w(l-w)}$ gives a linear relationship (Fig. 4B), with the slope scales with the ratio of viscosity over surface tension.