Figure 3.

Techniques for characterizing the condensed phase formed in 3D solution. A, DIC (left image) coupled with fluorescence imaging (middle and right images) of the phase droplets and multiple labeling of different components demonstrate their co-localization. B–D, dynamic properties of the condensed phase. E, fluorescence intensity–based absolute concentration estimation (adapted from Refs. 44, 45). A standard curve of fluorescence intensity to dye concentration is initially generated for calibration. Z direction scanning is performed to determine the proper focal plane for concentration estimation. At each Z stack, the fluorescence intensity distribution is plotted. Within the Z dimension of selected droplets, average fluorescence intensities are then compared across different layers. In a given system, the fluorescence intensity is constant regardless of the droplet size, and therefore the absolute protein concentration within a condensed phase can be calculated from the standard curve.