Liquid–liquid phase separation is ubiquitous in suspensions of nanoparticles, proteins and colloids. It has an important role in gel formation, protein crystallization and perhaps even as an organizing principle in cellular biology1,2. With a few notable exceptions3,4, liquid–liquid phase separation in bulk proceeds through the continuous coalescence of droplets until the system undergoes complete phase separation. But when colloids, nanoparticles or proteins are confined to interfaces, surfaces or membranes, their interactions differ fundamentally from those mediated by isotropic solvents5,6, and this results in significantly more complex phase behaviour7,8,9,10,11,12,13. Here we show that liquid–liquid phase separation in monolayer membranes composed of two dissimilar chiral colloidal rods gives rise to thermodynamically stable rafts that constantly exchange monomeric rods with the background reservoir to maintain a self-limited size. We visualize and manipulate rafts to quantify their assembly kinetics and to show that membrane distortions arising from the rods’ chirality lead to long-range repulsive raft–raft interactions. Rafts assemble into cluster crystals at high densities, but they can also form bonds to yield higher-order structures. Taken together, our observations demonstrate a robust membrane-based pathway for the assembly of monodisperse membrane clusters that is complementary to existing methods for colloid assembly in bulk suspensions14,15,16. They also reveal that chiral inclusions in membranes can acquire long-range repulsive interactions, which might more generally have a role in stabilizing assemblages of finite size13,17.
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We acknowledge discussions with R. B. Meyer. This work was supported by the US National Science Foundation (NSF-MRSEC-0820492, NSF-DMR-0955776 and NSF-MRI-0923057) and the Petroleum Research Fund (ACS-PRF 50558-DNI7). We acknowledge use of the Brandeis MRSEC optical microscopy facility.
The authors declare no competing financial interests.
This video shows coalescence of membranes composed of pure short rods (left) and pure long rods (right) as viewed in DIC imaging.
The long-short rod interface destabilizes to form rafts of well-defined size connected by thin bridges, which eventually break through thermal fluctuations. Scale bar, 2.56 μm. (MOV 1913 kb)
This video shows the fluorescence imaging time lapse of the intensity of rafts after they have been photo-bleached with a focused laser beam.
Some of the rafts have not been photo-bleached to highlight the difference between an unbleached and a photo-bleached raft. Short rods are fluorescently labeled and long rods are unlabeled, which causes the unbleached short-rod-rich rafts to appear bright and the short-rod-poor membrane background to appear dim. Scale bar, 2.7 μm. (MOV 2492 kb)
This video shows the time evolution of the polydisperse-sized rafts over a period of 19 hours as viewed in fluorescence imaging wherein only short rods are labeled (bright pixels).
Please note that at equilibrium, all rafts have nearly the same size. Using optical traps, a small fraction of rafts are broken into smaller rafts and others are merged into larger rafts (process not shown in the video). The three rafts seen in the video are marked by red, green and magenta circles, which are to highlight the three types of evolution observed. The raft marked by a red circle shrinks completely by losing all its rods into the background membrane. The rafts marked by green and magenta circles grow and shrink in size, respectively, with increasing time. Scale bar, 3.4 μm. (MOV 5899 kb)
Phase contrast and florescence images were acquired simultaneously and were subsequently overlaid. Longer rods are unlabeled. 1 out of 30,000 short rods are labeled and appear as isolated red spots. The phase contrast channel is false colored with rafts colored in yellow and the membrane background in green. The labeled rods diffuse within the membrane background. On reaching a raft interior, they reside in it with an average lifetime of a few minutes before eventually leaving the raft. Scale bar, 2.56 μm. (MOV 3740 kb)
Two optical plows composed of multiple light beams (each beam indicated as a red dot) push a raft pair into close proximity. Subsequently, optical plows are switched off and rafts move away from each other under the influence of repulsive interactions. (MOV 1816 kb)
This video shows fluorescence imaging time lapse of rafts in a membrane for three different long and short rod stoichiometric ratios at the same dextran concentration of 40mg/ml.
Short rods are fluorescently labeled and long rods are unlabeled. Rafts embedded in the host membrane exhibit a dilute gas-like structure at low number density of rafts. Short-range order sets in at intermediate raft densities. Rafts self-organize into a 2-D hexagonal lattice at the highest number density. Scale bar, 2.56 μm for the low and intermediate raft densities and 2.0 μm for the highest raft density. (MOV 6662 kb)
This video shows supra-clusters formed by stabilizing the thin liquid bridges between rafts in fluorescence imaging mode.
Short rods are fluorescently labeled and long rods are unlabeled. The video was made by combining three different image stacks that were acquired under the same experimental conditions. They are clearly demarcated in the video with thin long white lines. Various shapes of supra-clusters are observed depending on the number of rafts bound in the cluster. All Scale bars, 2.56 μm. (MOV 3174 kb)
This video shows fluorescence imaging time lapse of a “bead on a string” polymer-like chain assembled by connecting nine rafts into a chain.
The chain surprisingly maintains a preferential bond angle of 1100. Scale bar, 2.56 μm. (MOV 388 kb)
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Sharma, P., Ward, A., Gibaud, T. et al. Hierarchical organization of chiral rafts in colloidal membranes. Nature 513, 77–80 (2014). https://doi.org/10.1038/nature13694
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