(a) Scanning confocal microscopy images of the crowding sensor in 40% w/w PEG showing coacervate formation.1,2 Conditions: 10 mM NaPi, 100 mM NaCl, pH 7.4. Image dimensions: 32 x 32 µm. The fluorescent spots are not due to aggregation, because YFP excitation did not indicate self-quenching (Supplementary Figure 2). The crowding sensor partitions in the coacervates, which are highly crowded with PEG and induce FRET ratios (YFP/CFP) of >20. (b) Emission spectrum upon excitation of mCitrine at 515 nm at different % w/w PEG 6000. No difference between the spectra is observed, other than the experimental variation. Conditions: 10 mM NaPi, pH 7.4. NaCl was omitted to prevent coacervate formation.