Posing for a picture: vesicle immobilization in agarose gel

Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biological preparations for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 μm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small molecules, proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solutions, and complex biochemical reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resolution 3D images of GUVs.


S2. Tubulation and deformation of a GUV in the presence of 1% w/v agarose.
. A) Three-dimensional reconstruction of a POPC GUV immobilized in 1% (w/v) agarose. Note that the vesicle is non-spherical and slightly deformed by the agarose gel. B) Image of the upper surface of the GUV shown in A. The formed tubes are always directed to the outside. Scale bars: 5m.
S3. Size distribution of GUVs in the presence and absence of agarose. Figure S3. Vesicle size distributions from two typical batches of POPC GUVs in the presence (red, n = 118) and absence (black, n = 111) of agarose.

S4. Mechanical effects of agarose on GUVs
Recently, we showed that residual agarose present inside GUVs grown on hybrid films of agarose and lipids (following the protocol in Ref. 1 ) significantly affects vesicle relaxation dynamics and membrane pore lifetime 2 . In the experiments shown here, where agarose was present exclusively outside, no adverse effects on vesicle size and stability were observed ( Figure S3). In order to compare GUV mechanics in both conditions of agarose present exclusively in the vesicle exterior or interior, we followed the GUV response to electric fields. We applied a single and strong DC electric pulse to induce vesicle deformation and poration. In agarose-free aqueous solution, strong DC pulses deform and porate GUVs, after which the vesicle initial shape is restored with a characteristic relaxation time  relax and the formed macropores close with pore lifetime t pore 3 . Deformation is expressed as the aspect ratio of the vesicle semi-axes, a/b (see the inset in Figure S4). A typical GUV deformation/poration relaxation dynamic in agarose-free solution upon pulse application is shown in Figure S6 Figure S5. Effect of 0.5 % w/v agarose inside or outside GUVs exposed to electric pulses (3 kV/cm, 150 s). A), B) Sequences of two GUVs encapsulating agarose during application of a DC pulse, observed by phase contrast (A) and epifluorescence (B). The asterisk in A indicates the remaining GUV membrane. The spherical object next to it is the agarose mesh located inside the GUV before poration. The diameters of the initial GUVs in A and B are about 40 m. C) Confocal snapshots of a GUV dispersed in agarose and encapsulating 2.5 M sulforhodamine B before and after application of a DC pulse. The membrane is labeled with 0.5 mol% NBD-PE. Numbers correspond to time relative to the application of the pulse. The electric field direction is shown as an arrow. D) Fluorescence intensity of the membrane (green) and of sulforhodamine B (red) across the vesicle (same as in panel C and Figure 2C in the main text) before (light red) and 1 min after (dark red) pulse application, measured on the corresponding confocal images shown as insets. Bars: 20 m.
We next examined the response of immobilized GUVs (agarose only outside) to electric pulses.
The vesicles contained encapsulated sulforhodamine B to allow detecting leakage through macropores and possibly long-lasting submicron pores, if present. As evidenced in Figure S5C, strong pulses lead to membrane poration (see interrupted membrane contour in green), and allow release of some of the encapsulated content. Analysis of the sulforhodamine B fluorescence intensity inside the GUV shows a 7% decrease after macropore resealing (see Figure S5D). Importantly, contrary to vesicles encapsulating agarose where residual agarose molecules block pore closure ( Figures S5A and S5B, see also Ref. 2 ), the membrane here fully reseals and no long-term leakage is detected, suggesting that the agarose scaffold around the vesicle does not obstruct the membrane resealing even though increasing the pore lifetime (t pore ~ 160 ms). The overall vesicle deformation is stronger than that of vesicles encapsulating agarose, but weaker than that of vesicles in agarose-free medium. This outcome is understandable considering the gel-like environment of the agarose-immobilized GUVs, which does not allow for the vesicles to freely deform. The relaxation time of the immobilized vesicles is also slower than that of agarose-free vesicles,  relax ~ 1.4 s ( Figure S4). This finding was reproducible for all vesicles analyzed, and observed for vesicles containing 50 or 100 mol% of POPG lipids (not shown). Apparently, the vesicle deformation induced by the pulse induces rearrangement of the agarose mesh around it, which in return slows down the relaxation of the deformed vesicle.
In summary, these results demonstrate a different behavior of GUVs subjected to mechanical strain when agarose is present in their interior or exterior. While GUV integrity is lost when agarose is present inside (Figures S5A and S5B), membrane integrity is maintained in immobilized vesicles (agarose outside, Figure S5C and S5D) and only their relaxation and pore lifetime are slowed down because of the presence of the external agarose scaffold. The results broadly validate the mechanical confinement provided by agarose and the preservation of vesicle structural integrity, allowing reliable measurements to be performed on agarose-immobilized GUVs.   (eq. 2 in the main text and Ref. 4 ). The nominal radius (r n = 5 m), the effective radius (r e = 9 m) and the bleaching depth K are shown in blue. The inset shows the image used for the measurement. The effective radius was determined on bilayer patches on the cover slip surface, which resulted from GUVs that spontaneously ruptured and adhered to the glass surface in the presence of salts. This approach allowed better imaging of the bleached area. B) Typical FRAP recovery curve for a POPC GUV immobilized in 0.5% (w/v) agarose. F o and F ∞ are the fluorescence intensities in the first post-bleach image and after recovery, respectively, and t 1/2 is the time to reach F 1/2 = (F o + F ∞ )/2.

S9. Distribution of the number of DiI C18 molecules in the FCS volume.
Figure S10. Histogram of the number of DiI C18 molecules in the FCS volume. The curve is a Gaussian fit of the data. Only measurements with dye concentration in the range between the dashed vertical lines were considered.