A simple method to make, trap and deform a vesicle in a gel

Abstract

We present a simple method to produce giant lipid pseudo-vesicles (vesicles with an oily cap on the top), trapped in an agarose gel. The method can be implemented using only a regular micropipette and relies on the formation of a water/oil/water double droplet in liquid agarose. We characterize the produced vesicle with fluorescence imaging and establish the presence and integrity of the lipid bilayer by the successful insertion of [Formula: see text]-Hemolysin transmembrane proteins. Finally, we show that the vesicle can be easily mechanically deformed, non-intrusively, by indenting the surface of the gel.

Figure 1

(a–f) (upper row) Sketch of the double emulsion production method. Green/orange/blue colors stand for internal/oil/external phases, respectively. (bottom row) Bright field images. Scale bars = 500 $\mu$m. The produced double emulsion droplet in(f) sediments in the liquid agarose and eventually gets trapped as the agarose gels. Simultaneously, the surrounding oil shell creams at the top of the droplet and a lipid bilayer is zipped on the lower part. A few minutes later, the pseudo-vesicle has been formed and is trapped in the gel, as sketched in (g). Bottom panel: Fluorescence macroscope image of a pseudo-vesicle loaded with carboxyfluorescein trapped in an agarose gel. Scale bar = 200 $\mu$m.

Figure 2

(a) Composite confocal microscopy image of a pseudo-vesicle trapped in an agarose gel. The internal phase contains carboxyfluorescein (green), while the oil phase contains Nile Red (red). (b) Composite image obtained by adding 1wt% fluorescent NBD-PC in the lipid mixture (green) and an oil containing Nile Red (red). Yellow color = red + green channels. For clarity, the image has been smoothed with a 2 pixel radius Gaussian filter. Scale bars = 200 $\mu$m. (c) Normalized radial fluorescence intensity profile, averaged over N=8 pseudo-vesicles labelled as in (b). The green triangles (resp. red disks) show the fluorescent lipids channel (resp. fluorescent oil channel). Error bars are SE of data. $R=316\pm 24$ $\mu$m is the average pseudo-vesicle size. (d) Sketch of $\alpha$HL insertion in the pseudo-vesicle membrane, using $\alpha$HL loaded SUV. (e) Time evolution of the normalized contrast $\mathrm{\Gamma }(t)/\mathrm{\Gamma }(t=0)$, for $\alpha$HL loaded pseudo-vesicles (red disks, N=5) and control experiments without $\alpha$HL (black squares, N=5). Error bars are SD of data. The solid line is an exponential fit of the data $\mathrm{\Gamma }(t)/\mathrm{\Gamma }(t=0)=e^{-t/\tau }\approx 1-t/\tau$, with $\tau =5.9\pm 0.3.{10}^{4}s$.

Figure 3

(a) Sketch of the mechanical excitation setup. (b-d) Macroscope fluorescence images of pseudo-vesicles either relaxed ((b) Z=0) or deformed (Z=0.5 and 1 mm in (c,d), respectively). In (c) the red line is the fit of the contour of the lower part of the pseudo-vesicle with an ellipse. Scale bar = 400 $\mu$m. (e) Piston position Z(t). (f) Major (red) and minor (blue) axes of the fitted ellipse as a function of time, for a 1000 s long cyclic indentation. (g) Compressive stress $\sigma$ as a function of the ellipse’s eccentricity e. Grey crosses correspond to all data points from (f), black circles are averages within bins of eccentricity $\delta e$= 0.01. Error bars are SD of the data. The red doted line is a linear fit to the data. Inset: Normal force as a function of the piston indentation. The line is a linear fit $F=\kappa z$, from which the gel compression modulus can be deduced, $K_{\mathit{gel}}=\kappa H/S$, with H the height of the gel.