Quantification and statistical analysis

To quantify the recruitment of the fluorescent peptides at the surface of LDs, we used the radial angle profile plugin of ImageJ software. This plugin measures the average signal intensity along the perimeter of concentric circles. It results in a plot of the intensity profile of a circular object for various positions relative to its center. We chose the maximum intensity profile as a measurement of recruited peptide density. The same method was used to quantify the PL density covering oil droplet.

Due to experimental limitations, the DEVs used in this study were mainly imaged along their equatorial cross-section, an easier configuration when using micropipettes for manipulation. However, when measuring the fluorescence signal of aLDs at this equatorial level, we found a significant attenuation as compared with the signal measured at the apex of the droplet (Fig. S2 A). This is very likely due to the mismatch in optical index between the bulk water phase and the oil phase of the droplet (with 63× objective).

To investigate this attenuation effect, we imaged a single oil droplet homogeneously marked with histidine mCherry (Fig. S2 D). We noticed that the intensity of confocal fluorescence signal was varying along the z axis (Fig. S2 D, left). We quantified this signal modification with intensity-linescans at different z positions (see Fig. S2 D, right). The signal was maximum at the lower apex plane, i.e., bottom of the droplet, and almost dimmed just above the equatorial position (see third slice, Fig. S2 D). We reasonably assumed that this effect was due to the refraction of the laser light by the refractive index variation at the droplet–buffer interface. Indeed, when imaged at the lower apex, the light beam from the microscope objective is directly reflected back to the imaging system. On the contrary, when the droplet is imaged at or above the equatorial cross-section, part of the light has to pass through the oil phase of the droplet and is certainly disrupted by the optical index mismatch, leading to the observed signal attenuation (Fig. S2, A and D).

Therefore, the signal measured at the apex (not affected by the optical index mismatch) is to be considered as the correct measurement of the fluorescent signal on the droplet. On the contrary, to get the real signal on the droplet at the equatorial cross-section, the measured signal has to be corrected.

To quantify the signal attenuation at the equatorial cross-section, we imaged various DEV at the equatorial cross-section and the apex. We normalized the droplet signal by the bilayer signal (Fig. S2 B) and compared the normalized signal measured at the apex and the equatorial cross-section. We found an average of 2.67 times less signal at the equatorial cross-section as compared with the apex.

We thus defined a correcting optical parameter β as the apex normalized signal divided by the equatorial cross-section normalized signal (Fig. S2 C), which was therefore equal to 2.67. To properly evaluate the fluorescence on aLDs imaged at the equatorial cross-section, the measured signal then had to be multiplied by the optical parameter β = 2.67.

As some of the DEVs used in this study were made with SQ oil droplets, we performed similar experiments on such systems and found comparable results (Fig. S2 E). The fluorescence signal attenuation between apex (focal plane 1) and equatorial cross-section (focal plane 2) was found at 2.7 (Fig. S2 E, right), a value comparable to those obtained with TO droplets in Fig. S2 B.

The statistical comparisons were made using a nonparametric t test (GraphPad Prism 7.0a; ***, P < 0.0001; **, P < 0.001; *, P < 0.01). Unless mentioned, all values shown in the text and figures are mean ± SD.