Individual Bilayers Research Articles

Stacking-order independent inter-layer charge transfer in MBE-grown MoSe2 and WSe2 heterostructures

MoSe2/WSe2 heterostructure is gaining significant research interest due to its unique electrical and optical properties, making them suitable for various advanced applications. This work focuses on inter-layer charge transfer effects on MBE-grown MoSe2 and WSe2 heterostructures. Bilayer (BL)-MoSe2/BL-WSe2 and BL-WSe2/BL-MoSe2 have been grown over a c-plane sapphire substrate using molecular beam epitaxy (MBE). Different characterization techniques including Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and reflection high-energy electron diffraction (RHEED) confirm the phase, composition, surface morphology, and crystalline quality of the films, respectively. The band diagram using Ultraviolet-visible (UV-Vis) spectroscopy, XPS core level spectra, XPS valence band spectra and surface work function shows that individual MoSe2 and WSe2 BLs grown over c-plane sapphire are n-type and p-type semiconductors, respectively. This study shows that when the individual BLs form MoSe2/WSe2 and WSe2/MoSe2 heterostructures, a unidirectional electron transfer occurs from the WSe2 to MoSe2, which is independent of WSe2 and MoSe2 stacking order. As a result, the Fermi level position changes for both the materials in their heterostructures, which makes MoSe2 more n-type semiconductor and WSe2 more p-type semiconductor. MoSe2/WSe2 and WSe2/MoSe2 heterostructures show a type II band alignment with a conduction band offset of 0.57 eV and a valance band offset of 0.51 eV These findings contribute to a deeper understanding of the interfacial charge transfer effects and design of MoSe2 and WSe2 heterostructures for potential device applications.

Energy band engineering in GaS/InS and GaSe/InS van der Waals bilayers by interlayer stacking design and applied vertical electric field - An ab-initio theoretical calculation based approach

In this work, for the first time, an extensive theoretical investigation of the structural and electronic properties of the van der Waals (vdW) bilayer heterostructures of two-dimensional (2D) Gallium Sulphide (GaS)/Indium Sulphide (InS) and Gallium Selenide (GaSe)/Indium Sulphide (InS), is performed using density functional theory calculation. In this context, the effects of natural and artificial interlayer stacking configurations, and externally applied out-of-plane direction electric field are emphasized. The structural, thermal, and dynamic stabilities of individual bilayers are assessed from the cohesive energy/interlayer interaction energy, phonon spectrum, and molecular dynamics evolution, respectively. The effects of the vdW bilayer environment on the in-plane structural parameters like lattice vector, bond length, and bond angle of the constituent monolayers are comprehensively investigated. The electronic properties are estimated from the band edge energy, the density of states, energy bandgap, and interlayer energy band alignment, which are systematically analysed from the interlayer interactions in terms of interlayer distance, out-of-plane charge distribution, and interlayer charge transfer. The key findings demonstrate that the interlayer stacking engineering and applied out-of-plane electric field can induce unique and tailor-made modulations in the overall electronic properties of GaS/InS and GaSe/InS bilayer, which can be exploited in favour of electronic/optoelectronic applications.

Leaflet Tensions Control the Spatio-Temporal Remodeling of Lipid Bilayers and Nanovesicles.

Biological and biomimetic membranes are based on lipid bilayers, which consist of two monolayers or leaflets. To avoid bilayer edges, which form when the hydrophobic core of such a bilayer is exposed to the surrounding aqueous solution, a single bilayer closes up into a unilamellar vesicle, thereby separating an interior from an exterior aqueous compartment. Synthetic nanovesicles with a size below 100 nanometers, traditionally called small unilamellar vesicles, have emerged as potent platforms for the delivery of drugs and vaccines. Cellular nanovesicles of a similar size are released from almost every type of living cell. The nanovesicle morphology has been studied by electron microscopy methods but these methods are limited to a single snapshot of each vesicle. Here, we review recent results of molecular dynamics simulations, by which one can monitor and elucidate the spatio-temporal remodeling of individual bilayers and nanovesicles. We emphasize the new concept of leaflet tensions, which control the bilayers' stability and instability, the transition rates of lipid flip-flops between the two leaflets, the shape transformations of nanovesicles, the engulfment and endocytosis of condensate droplets and rigid nanoparticles, as well as nanovesicle adhesion and fusion. To actually compute the leaflet tensions, one has to determine the bilayer's midsurface, which represents the average position of the interface between the two leaflets. Two particularly useful methods to determine this midsurface are based on the density profile of the hydrophobic lipid chains and on the molecular volumes.

Comparative analysis of strain engineering on the electronic properties of homogenous and heterostructure bilayers of MoX2 (X = S, Se, Te)

In this work, for the first time, the comparative electronic properties of homogenous and van der Waals (vdW) heterostructure bilayers of Molybdenum Dichalcogenides, MoX2 (X = S, Se, Te) is investigated using first-principle calculations with the emphasis on their response to applied biaxial mechanical strain. The dynamical, thermal, and energetical stability of the vdW bilayers are assessed in detail. Next, a comprehensive analysis of energy band structure with the atomic orbital projections of band edges has been performed for individual bilayers. In this context, the energy of different conduction and valence band edges are studied within a range of 5% biaxial compressive (BC) to 5% biaxial tensile (BT) strain. The applied strain-dependent band edge energy modulations are extensively analysed based on the change in structural properties and thereby the strength of in-plane and interlayer atomic orbital couplings. The analysis is further extended to correlate the variations in energy bandgaps and density of states (DOS) in different bilayers with applied strain. The results indicate that, unlike homogenous bilayers, the vdW bilayers demonstrate distinct electronic properties in their relaxed configuration. Specifically, the MoSe2/MoTe2, MoS2/MoSe2, and MoS2/MoTe2 exhibit small direct bandgap, moderate indirect bandgap, and metallic/semimetallic nature, respectively. Furthermore, distinctly different modulations in conduction band minima (CBM) and valence band maxima (VBM) positions in the Brillouin zone are observed with applied strain for individual vdW bilayers in contrast to almost similar nature of variations in CBM/VBM positions of homogenous bilayers. This leads to characteristic nonlinear variations in energy bandgap and distinct DOS modulations for individual vdW bilayers under applied strain. The key findings indicate that the suitably strained VdW bilayer MoX2 are highly promising materials for a plethora of electronic and optoelectronic applications.

Magnetron sputtered NiAl/TiBx multilayer thin films

Transition metal diboride-based thin films are currently receiving strong interest in fundamental and applied research. Multilayer thin films based on transition metal diborides are, however, not yet explored in detail. This study presents results on the constitution and microstructure of multilayer thin films composed of TiBx and the intermetallic compound NiAl. Single layer NiAl and TiBx and NiAl/TiBx multilayer thin films with a variation of the individual layer thickness and bilayer period were deposited by D.C. and R.F. magnetron sputtering on silicon substrates. The impact of the operation mode of the sputtering targets on the microstructure of the thin films was investigated by detailed compositional and structural characterization. The NiAl single layer thin films showed an operation mode-dependent growth in a polycrystalline B2 CsCl structure with a cubic lattice with and without preferred orientation. The TiBx single layer thin films exhibited an operation mode independent crystalline structure with a hexagonal lattice and a pronounced (001) texture. These TiBx layers were significantly Ti-deficient and showed B-excess, resulting in stoichiometry in the range TiB2.64–TiB2.72. Both thin film materials were deposited in a regime corresponding with zone 1 or zone T in the structure zone model of Thornton. Transmission electron microscopy studies revealed, however, very homogeneous, dense thin-film microstructures, as well as the existence of dislocation lines in both materials. In the multilayer stacks with various microscale and nanoscale designs, the TiBx layers grew in a similar microstructure with (001) texture, while the NiAl layers were polycrystalline without preferred orientation in microscale design and tended to grow polycrystalline with (211) preferred orientation in nanoscale designs. The dislocation densities at the NiAl/TiBx phase boundaries changed with the multilayer design, suggesting more smooth interfaces for multilayers with microscale design and more disturbed, strained interfaces in multilayers with nanoscale design. In conclusion, the volume fraction of the two-layer materials, their grain size and crystalline structure, and the nature of the interfaces have an impact on the dislocation density and ability to form dislocations in these NiAl/TiBx-based multilayer structures.

Encapsulated droplet interface bilayers as a platform for high-throughput membrane studies.

Whilst it is highly desirable to produce artificial lipid bilayer arrays allowing for systematic high-content screening of membrane conditions, it remains a challenge due to the combined requirements of scaled membrane production, simple measurement access, and independent control over individual bilayer experimental conditions. Here, droplet bilayers encapsulated within a hydrogel shell are output individually into multi-well plates for simple, arrayed quantitative measurements. The afforded experimental throughput is used to conduct a 2D concentration screen characterising the synergistic pore-forming peptides Magainin2 and PGLa. Maximal enhanced activity is revealed at equimolar peptide concentrations via a membrane dye leakage assay, a finding consistent with models proposed from NMR data. The versatility of the platform is demonstrated by performing in situ electrophysiology, revealing low conductance pore activity (∼15 to 20 pA with 4.5 pA sub-states). In conclusion, this array platform addresses the aforementioned challenges and provides new and flexible opportunities for high-throughput membrane studies. Furthermore, the ability to engineer droplet networks within each construct paves the way for “lab-in-a-capsule” approaches accommodating multiple assays per construct and allowing for communicative reaction pathways.

Magnetic properties of the intercalated compoundLu2Fe3O7

Complex spin and charge order in the frustrated Fe/O bilayers of rare-earth ferrites $R$Fe${}_{2}$O${}_{4}$ and proposed multiferroicity for $R$ = Lu have attracted a lot of interest. The authors focus on $i\phantom{\rule{0}{0ex}}n\phantom{\rule{0}{0ex}}t\phantom{\rule{0}{0ex}}e\phantom{\rule{0}{0ex}}r\phantom{\rule{0}{0ex}}c\phantom{\rule{0}{0ex}}a\phantom{\rule{0}{0ex}}l\phantom{\rule{0}{0ex}}a\phantom{\rule{0}{0ex}}t\phantom{\rule{0}{0ex}}e\phantom{\rule{0}{0ex}}d$ Lu${}_{2}$Fe${}_{3}$O${}_{7}$, with additional Fe/O single layers inserted between the bilayers. Macroscopic magnetization measurements, polarized neutron scattering, and x-ray magnetic circular dichroism indicate that spin-charge coupling and the local spin arrangement in each individual bilayer is the same as in the nonintercalated compound, with the Fe spins in the single layers giving rise to an additional paramagneticlike contribution.

Mechanism of Cell Penetration by Permeabilization of Late Endosomes: Interplay between a Multivalent TAT Peptide and Bis(monoacylglycero)phosphate

Many cellular delivery reagents enter the cytosolic space of cells by escaping the lumen of endocytic organelles and, more specifically, late endosomes. The mechanisms involved in endosomal membrane permeation remain largely unresolved, which impedes the improvement of delivery agents. Here, we investigate how 3TAT, a branched analog of the cell-penetrating peptide (CPP) TAT, achieves the permeabilization of bilayers containing bis(monoacylglycero)phosphate (BMP), a lipid found in late endosomes. We establish that the peptide does not induce the leakage of individual lipid bilayers. Instead, leakage requires contact between membranes. Peptide-driven bilayer contacts lead to fusion, lipid mixing, and, critically, peptide encapsulation within proximal bilayers. Notably, this encapsulation is a distinctive property of BMP that explains the specificity of CPP's membrane leakage activity. These results therefore support a model of cell penetration that requires both BMP and the vicinity between bilayers, two features unique to BMP-rich and multivesicular late endosomes.

Mechanism of Cell Penetration by Permeabilization of Late Endosomes: Interplay between a Multivalent TAT-Like Cell-Penetrating Peptide and the Lipid Bis(Monoacylglycerol)Phosphate

Many cellular delivery reagents enter the cytosolic space of cells by escaping the lumen of endocytic organelles and, more specifically, late endosomes. The mechanisms involved in endosomal membrane permeation remain largely unresolved, which impedes the improvement of delivery agents. Herein, we investigate how 3TAT, a branched analog of the cell-penetrating peptide (CPP) TAT, achieves the permeabilization of bilayers containing bis(monoacylglycerol)phosphate (BMP), a lipid found in late endosomes. We establish that the peptide does not induce the leakage of individual lipid bilayers. Instead, leakage requires contact between membranes. Peptide-driven bilayer contacts lead to fusion, lipid mixing, and, critically, peptide encapsulation within proximal bilayers. Notably, this encapsulation is a distinctive property of BMP that explains the specificity of the CPP’s membrane leakage activity. These results therefore support a novel model of cell penetration that requires both BMP and the vicinity between bilayers, two features unique to BMP-rich and multivesicular late endosomes.

Gap Opening in Twisted Double Bilayer Graphene by Crystal Fields.

Crystal fields occur due to a potential difference between chemically different atomic species. In van der Waals heterostructures such fields are naturally present perpendicular to the planes. It has been realized recently that twisted graphene multilayers provide powerful playgrounds to engineer electronic properties by the number of layers, the twist angle, applied electric biases, electronic interactions, and elastic relaxations, but crystal fields have not received the attention they deserve. Here, we show that the band structure of large-angle twisted double bilayer graphene is strongly modified by crystal fields. In particular, we experimentally demonstrate that twisted double bilayer graphene, encapsulated between hBN layers, exhibits an intrinsic band gap. By the application of an external field, the gaps in the individual bilayers can be closed, allowing to determine the crystal fields. We find that crystal fields point from the outer to the inner layers with strengths in the bottom/top bilayer [Formula: see text] = 0.13 V/nm ≈ [Formula: see text] = 0.12 V/nm. We show both by means of first-principles calculations and low energy models that crystal fields open a band gap in the ground state. Our results put forward a physical scenario in which a crystal field effect in carbon substantially impacts the low energy properties of twisted double bilayer graphene, suggesting that such contributions must be taken into account in other regimes to faithfully predict the electronic properties of twisted graphene multilayers.

Probing Exfoliated Graphene Layers and Their Lithiation with Microfocused X-rays.

X-ray diffraction is measured on individual bilayer and multilayer graphene single-crystals and combined with electrochemically induced lithium intercalation. In-plane Bragg peaks are observed by grazing incidence diffraction. Focusing the incident beam down to an area of about 10 μm × 10 μm, individual flakes are probed by specular X-ray reflectivity. By deploying a recursive Parratt algorithm to model the experimental data, we gain access to characteristic crystallographic parameters of the samples. Notably, it is possible to directly extract the bi/multilayer graphene c-axis lattice parameter. The latter is found to increase upon lithiation, which we control using an on-chip peripheral electrochemical cell layout. These experiments demonstrate the feasibility of in situ X-ray diffraction on individual, micron-sized single crystallites of few- and bilayer two-dimensional materials.

A New Computational Method for Membrane Compressibility: Bilayer Mechanical Thickness Revisited

Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity (Kc) and area compressibility (Ka). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. While computational tools such as Molecular Dynamics (MD) simulations can provide alternative means to estimate Ka values, current approaches suffer significant technical limitations. Here, we present a novel computational framework that allows for a direct estimation of Ka values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives Ka from real-space analysis of local thickness fluctuations sampled in MD simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid composition and sizes, simulated at different temperatures. The calculated bilayer compressibility moduli agree with values estimated previously from experiments and those obtained from a standard computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model (PBM) and confirm the PBM's predicted linear relationship with proportionality coefficient of 24 using elastic parameters calculated from the simulation trajectories. The robustness of the results that emerge from the new method allows us to revisit the origins of the bilayer mechanical (compressible) thickness and in particular, its dependence on acyl chain unsaturation and the presence of cholesterol.

A New Computational Method for Membrane Compressibility: Bilayer Mechanical Thickness Revisited

Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity (κC) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions, and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. Although computational tools such as molecular dynamics simulations can provide alternative means to estimate KA values, current approaches suffer significant technical limitations. Here, we present a novel, to our knowledge, computational framework that allows for a direct estimation of KA values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives KA from real-space analysis of local thickness fluctuations sampled in molecular dynamics simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid compositions and sizes, simulated at different temperatures. The calculated bilayer compressibility moduli agree with values estimated previously from experiments and those obtained from a standard computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model and confirm the polymer brush model’s predicted linear relationship with proportionality coefficient of 24, using elastic parameters calculated from the simulation trajectories. The robustness of the results that emerge from the method allows us to revisit the origins of the bilayer mechanical (compressible) thickness and in particular its dependence on acyl-chain unsaturation and the presence of cholesterol.

Invited Article: Heterodyne dual-polarization epi-detected CARS microscopy for chemical and topographic imaging of interfaces

Coherent Raman Scattering (CRS) has emerged in the last decade as a powerful multiphoton microscopy technique offering chemically specific label-free imaging in real time with high three-dimensional spatial resolution. Many technical realizations of CRS microscopy have been proposed to remove, suppress, or account for the non-resonant background in the nonlinear susceptibility which complicates spectral analysis and reduces image contrast. Here, we demonstrate coherent anti-Stokes Raman scattering microscopy using a dual-polarization balanced heterodyne detection in epi-geometry (eH-CARS), providing background-free chemically specific image contrast for nanoparticles and interfaces, shot-noise limited detection, and phase sensitivity. We show the sensitivity and selectivity of eH-CARS in comparison with forward CARS and stimulated Raman scattering on polystyrene beads in agarose gel. As an important biologically relevant application, we demonstrate eH-CARS imaging of individual lipid bilayers with high contrast and topographic sensitivity.

Evaluation of spontaneous superlattice ordering in MOCVD grown AlxGa1-xAs epilayer on GaAs (100) using X-ray reflectivity and rocking curve analysis

Spontaneous superlattice structure in few monolayer length scale has been observed very recently in AlxGa1-xAs epilayer on (001) GaAs substrate grown by Metal Organic Chemical Vapor Deposition. Here we have reported a detailed study of this structure using high resolution x-ray reflectivity (XRR) and rocking curve (XRC) analysis and tried to extract composition modulation in the superlattice. A model-independent Distorted Wave Born Approximation (DWBA) has been applied to the XRR results to successfully obtain the electron density profile of the structure from which the compositional information could be extracted. The XRR profile of the AlxGa1-xAs sample (x = 0.17) showed sharp equidistant peaks giving clear evidence of superlattice ordering in the epilayer. Simulation of the XRR results showed a bilayer segregation with alternating Al composition of 0.1 and 0.4 of thickness 20 and 8 Å, respectively with interfacial thicknesses of 12 Å on both sides, giving a periodicity of 52 Å. The cross-sectional transmission electron microscopic (XTEM) image also showed similar length scale. A kinematical model was then used successfully to fit the sharp peaks observed in the XRC using the characteristic length and compositional parameters obtained from the XRR simulation. These results also showed that the individual bilayers were of single crystalline zinc blende structures. Both the simulation techniques were first applied to a test structure of AlAs/GaAs multilayer of known thickness before applying the same to the spontaneous superlattice to validate the model.

Punching Membranes: How Lipid Bilayers Withstand and Propagate Mechanical Load

Mechanical perturbations are ubiquitous in living cells and many biological functions are strongly dependent on the mechanical response of lipid membranes. We asked how lipid bilayers withstand and also propagate high mechanical load using large-scale atomistic and coarse-grained Molecular Dynamics simulations. We perforated stacked lipid bilayers using a spherical indenter, closely mimicking novel force spectroscopy techniques that capture the fracture of individual membranes, while avoiding substrate effects by probing stacks of model bilayers. We observe a step-wise fracture of individual bilayers in close agreement with experiments. Dwell times of the physiologically more relevant free standing bilayers are lower than those of stacked bilayers. Interestingly, the rupture mechanism as well as the dwell time distribution depend on the nature of the indenter: Only a hydrophilic intender yields the expected log-normal distribution by inducing a pore-growth mechanism. Upon subjecting the bilayer to a longitudinal rather than a perpendicular perturbation, we observe the propagation of a mechanical pulse through the lipid bilayer with the experimentally known speed of sound of ∼1 km/s, again in both atomistic and coarse-grained systems. The computed dispersion follows proposed continuum visco-elastic models and is remarkably low, allowing stress propagation over tens of nanometers before damping Our results provide a better understanding of membrane nanomechanics and indicate that membranes play a role as efficient wave guides for signalling through the cell.

In-plane molecular organization of hydrated single lipid bilayers: DPPC:cholesterol.

Understanding the physical properties of cholesterol-phospholipid systems is essential to gain a better knowledge of the function of each membrane constituent. We present a novel, simple and user-friendly setup that allows for the straightforward grazing incidence X-ray diffraction characterization of hydrated individual supported lipid bilayers. This configuration minimizes the scattering from the liquid and allows the detection of the extremely weak diffracted signal of the membrane, enabling the differentiation of the coexisting domains in DPPC:cholesterol single bilayers.

Quasi 3D polymerization in C60 bilayers in a fullerene solvate

The polymerization of fullerenes has been an interesting topic for almost three decades. A rich polymeric phase diagram of C60 has been drawn under a variety of pressure-temperature conditions. However, only linear or perpendicular linkages of C60 are found in the ordered phases. Here we used a unique bilayer structural solvate, C60·1,1,2-trichloroethane (C60·1TCAN), to generate a novel quasi-3D C60 polymer under high pressure and/or high temperature. Using Raman, IR spectroscopy and X-ray diffraction, we observe that the solvent molecules play a crucial role in confining the [2 + 2] cycloaddition bonds of C60s forming in the upper and lower layers alternately. The relatively long distance between the two bilayers restricts the covalent linkage extended in a single individual bilayer. Our studies not only enrich the phase diagram of polymeric C60, but also facilitate targeted design and synthesis of unique C60 polymers.

The Nanomechanics of Lipid Multibilayer Stacks Exhibits Complex Dynamics.

The nanomechanics of lipid membranes regulates a large number of cellular functions. However, the molecular mechanisms underlying the plastic rupture of individual bilayers remain elusive. This study uses force clamp spectroscopy to capture the force-dependent dynamics of membrane failure on a model diphytanoylphosphatidylcholine multilayer stack, which is devoid of surface effects. The obtained kinetic measurements demonstrate that the rupture of an individual lipid bilayer, occurring in the bilayer parallel plane, is a stochastic process that follows a log-normal distribution, compatible with a pore formation mechanism. Furthermore, the vertical individual force-clamp trajectories, occurring in the bilayer orthogonal bilayer plane, reveal that rupturing process occurs through distinct intermediate mechanical transition states that can be ascribed to the fine chemical composition of the hydrated phospholipid moiety. Altogether, these results provide a first description of unanticipated complexity in the energy landscape governing the mechanically induced bilayer rupture process.

Investigation of Transbilayer Coupling in Gel-Fluid Asymmetric Lipid Vesicles

Biological membranes display an asymmetric lipid distribution along the bilayer normal. Membrane asymmetry is also thought to affect the bilayer and the individual bilayer leaflet thicknesses. Recently, protocols enabling the preparation and characterization of stress-free, free-floating asymmetric vesicles (aLUVs) via a cyclodextrin mediated exchange were developed [1]. Here, we report of aLUVs studies with gel and fluid phase bilayer leaflets using small- and wide-angle x-ray scattering (SWAXS), and small-angle neutron scattering (SANS). Together, these techniques allow for obtaining the structural details of the two membrane leaflets. No hydrocarbon chain correlation peak was observed in the wide-angle regime of DPPC/POPC aLUVs at room temperature, consistent with an earlier observation of the partial fluidization of gel DPPC through coupling to the inner, fluid POPC leaflet [1]. On the other hand, POPE/POPC aLUVs, below the melting temperature of pure POPE, showed a WAXS-peak typical of hydrocarbons packed in a 2D hexagonal lattice. This coupling between the two bilayer leaflets manifested itself as through a small increase in area per lipid. However, our studies also showed that the underlying energetic contributions are insufficient to break up the hydrogen bond-stabilizing network formed by PE headgroups.This work is supported by the Austrian Science Fund FWF, Project No.P27083-B20 (to G.P.).[1] Heberle, F. A., Marquardt, D., Doktorova, M., Geier, B., Standaert, R. F., Heftberger, P., Kollmitzer, B., Nickels, J. D., Dick, R. A., Feigenson, G. W. et al. (2016). Langmuir, 32(20), 5195(5200).