Domains In Model Membranes Research Articles

Inimitable Impacts of Ceramides on Lipid Rafts Formed in Artificial and Natural Cell Membranes

Ceramide is the simplest precursor of sphingolipids and is involved in a variety of biological functions ranging from apoptosis to the immune responses. Although ceramide is a minor constituent of plasma membranes, it drastically increases upon cellular stimulation. However, the mechanistic link between ceramide generation and signal transduction remains unknown. To address this issue, the effect of ceramide on phospholipid membranes has been examined in numerous studies. One of the most remarkable findings of these studies is that ceramide induces the coalescence of membrane domains termed lipid rafts. Thus, it has been hypothesised that ceramide exerts its biological activity through the structural alteration of lipid rafts. In the present article, we first discuss the characteristic hydrogen bond functionality of ceramides. Then, we showed the impact of ceramide on the structures of artificial and cell membranes, including the coalescence of the pre-existing lipid raft into a large patch called a signal platform. Moreover, we proposed a possible structure of the signal platform, in which sphingomyelin/cholesterol-rich and sphingomyelin/ceramide-rich domains coexist. This structure is considered to be beneficial because membrane proteins and their inhibitors are separately compartmentalised in those domains. Considering the fact that ceramide/cholesterol content regulates the miscibility of those two domains in model membranes, the association and dissociation of membrane proteins and their inhibitors might be controlled by the contents of ceramide and cholesterol in the signal platform.

Direct Imaging of Lipid Domains in Nanoscale Vesicles by cryoEM

Direct imaging of membrane domains has provided evidence necessary to answer a wide range of scientific questions; direct imaging has been used to prove that vacuole membranes phase separate in living yeast cells and to show that current theories of modulated phases are incomplete. Many deep questions about membrane domains remain extremely challenging to answer because our current arsenal of direct imaging techniques is incomplete. For example, the existence of sub-micron domains in model membranes (and in some cases, the cell plasma membrane) has been inferred by indirect methods such as FRET, neutron scattering, and NMR. In cases where indirect methods require assumption-dependent models, it is important to verify the results directly with microscopy. To overcome current limitations, new methods of imaging sub-micron membrane domains are needed. Here, we present two novel cryo-EM methods of imaging sub-micron lipid domains in ternary model membranes. The first method employs a monovalent probe that is both fluorescent and electron-dense, enabling direct, and model-free comparisons for a single vesicle analyzed by both fluorescence microscopy and electron microscopy. The second method is entirely label-free; it identifies domains that are thicker than the surrounding membrane. To validate this method, we leverage existing phase diagrams (obtained through fluorescence microscopy) and AFM data to identify compositions with a membrane thickness difference between the Lo phase and Ld phase of ≥ 1 nm. We benchmark our techniques against known phase diagrams and demonstrate that our new methods accurately quantify the area fractions of each phase.

Partitioning of Lipids between Domains in Model Membranes Studied by Coarse-Grained MD Simulations

Partitioning of Lipids between Domains in Model Membranes Studied by Coarse-Grained MD Simulations

Preferential binding and re-organization of nanoscale domains on model lipid membranes by pore-forming toxins: insight from STED-FCS

Potential nanodomains in cellular membranes are widely believed to be targeted by proteins and other biomolecules to enable execution of critical cellular functions. However, characterization of these nanodomains remains elusive, primarily due to the diffraction limit of a conventional optical microscope. Herein using super-resolution STED microscopy coupled with fluorescence correlation spectroscopy (STED-FCS), we provide experimental evidence of lipid nanodomains present in model membranes comprised of phosphocholine-cholesterol binary mixture, and its reorganization induced by pore-forming toxins (PFTs). In this study, we used two different types of PFTs, namely α-PFT (cytolysin A) and β-PFT (listeriolysin O), that preferentially associate as well as reorganize cholesterol-containing saturated and unsaturated phosphocholine membranes. The emergence of nanodomains due to the lipid–lipid and lipid-PFT interactions was quantified at a length scales of ~50–150 nm using FCS diffusion law enabled by variation of spot sizes in the super-resolution STED microscopy. Our results shed light on the usefulness of super-resolution microscopy to quantify the underlying nanoscale domains in model membranes with implications for a wide variety of membrane-mediated cellular events observed in real cell membranes.

The Alzheimer's disease amyloid-β peptide affects the size-dynamics of raft-mimicking Lo domains in GM1-containing lipid bilayers.

Alzheimer's disease (AD) is characterized by the overproduction of the amyloid-β peptide (Aβ) which forms fibrils under the influence of raft microdomains containing the ganglioside GM1. Raft-mimicking artificial liquid ordered (Lo) domains containing GM1 enhance amyloid-β polymerization. Other experiments suggest that Aβ binds preferably to the non-raft liquid disordered (Ld) phase rather than to the Lo phase in the presence of GM1. Here, the interaction of Aβ(1-42) with GM1-containing biphasic Lo-Ld giant vesicles was investigated. Fluorescence colocalisation experiments confirm that Aβ(1-42) binds preferentially to the Ld phase. The effect of Aβ(1-42) on the Lo-Ld size dynamics was studied using photoinduced spinodal decomposition which mimics the nanodomain-microdomain raft coalescence. Aβ affects the kinetics of the coarsening phase and the size of the resulting microdomains. The effect depends on which phase is in a majority: when the Lo microdomains are formed inside an Ld phase, their growth rate becomes slower and their final size smaller in the presence of Aβ(1-42), whereas when the Ld microdomains are formed inside an Lo phase, the growth rate becomes faster and the final size larger. Fluorimetric measurements on large vesicles using the probe Laurdan indicate that Aβ(1-42) binding respectively increases or decreases the packing of the Ld phase in the presence or absence of GM1. The differential effects of Aβ on spinodal decomposition are accordingly interpreted as resulting from distinct effects of the peptide on the Lo-Ld line tension modulated by GM1. Such modulating effect of Aβ on domain dynamics could be important for lipid rafts in signaling disorders in AD as well as in Aβ fibrillation.

Permeation pathways through lateral domains in model membranes of skin lipids.

An understanding of how molecules permeate the complex lipid matrix of the stratum corneum (SC) skin barrier is important for transdermal drug delivery, preventing the adsorption of toxic chemicals and tackling skin diseases. In this paper we present atomistic molecular dynamics simulations of skin-lipid bilayers composed of ceramides, cholesterol (CHOL) and free fatty acids at different lipid compositions and levels of hydration and investigate both perpendicular and lateral permeation pathways through the systems. We show that in fully hydrated bilayers the lipids are heterogeneously distributed, with CHOL-rich domains emerging spontaneously during the simulations. Potential of mean constraint force calculations reveal that the most favourable permeation pathway for water in the direction normal to the bilayer is through a CHOL-rich region, probably due to the disordering effect of CHOL on lipids in the gel-phase. In systems with a low water content (akin to real skin) we find that rather than forming continuous layers, water forms flattened ellipsoid-shaped pools between the lipid headgroups, which are separated by dry regions. This implies that there is no continuous aqueous lateral pathway in the SC and may help to explain why skin is such an effective barrier. We propose that the most probable permeation pathway for a small polar molecule consists of hopping from the headgroup region of one bilayer to the next via a dry region, followed by permeation along the bilayer normal through a CHOL-rich region to the centre of the bilayer where it can diffuse laterally in the lower-density lipidic environment before encountering another CHOL-rich region through which it can exit the bilayer.

Cholesterol Bilayer Domains in the Eye Lens Health: A Review

The most unique biochemical characteristic of the eye lens fiber cell plasma membrane is its extremely high cholesterol content, the need for which is still unclear. It is evident, however, that the disturbance of Chol homeostasis may result in damages associated with cataracts. Electron paramagnetic resonance methods allow discrimination of two types of lipid domains in model membranes overloaded with Chol, namely, phospholipid-cholesterol domains and pure Chol bilayer domains. These domains are also detected in human lens lipid membranes prepared from the total lipids extracted from lens cortices and nuclei of donors from different age groups. Independent of the age-related changes in phospholipid composition, the physical properties of phospholipid-Chol domains remain the same for all age groups and are practically identical for cortical and nuclear membranes. The presence of Chol bilayer domains in these membranes provides a buffering capacity for cholesterol concentration in the surrounding phospholipid-Chol domains, keeping it at a constant saturating level and thus keeping the physical properties of the membrane consistent with and independent of changes in phospholipid composition. It seems that the presence of Chol bilayer domains plays an integral role in the regulation of cholesterol-dependent processes in fiber cell plasm membranes and in the maintenance of fiber cell membrane homeostasis.

Phases and Fluctuations in Biological Membranes

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase separated domains in model membranes. However, this concept remains controversial due to the difficulty in directly observing these domains in intact cells. Here, we visualize ordered and disordered phase-like domains in intact B cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, establish that ordered domains are local environments that favor tyrosine phosphorylation, and present a minimal and predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling as well as a plausible mechanism of BCR activation via receptor clustering. More generally, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that may broadly impact signal transduction.

Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

Physiochemical Properties of Aluminum Adjuvants Elicit Differing Reorganization of Phospholipid Domains in Model Membranes.

Most vaccines contain aluminum adjuvants; however, their exact mechanism of action remains unclear. A novel mechanism by Shi and colleagues proposes aluminum adjuvants may enhance immune activation by binding and reorganizing lipids that are key components of lipid rafts. To better understand the specificity of interaction between aluminum adjuvants and the cell membrane lipids, we present a biophysical study of lipid domain clustering in simple model phospholipid monolayers containing dipalmitoyl-phosphatidylcholine (DPPC) and dioleoyl-phosphatidylcholine (DOPC) exposed to two aluminum adjuvants, Alhydrogel and Adju-Phos. Surface pressure measurements and fluorescence microscopy images verified aluminum adjuvant-induced increase in lipid domain size, even in the key lipid raft components. Additionally, adjuvant induced lipid clustering differed based on the physicochemical properties of the adjuvants. Alhydrogel appeared to reduce monolayer compressibility and insert into the monolayer, while Adju-Phos induced more significant changes in domain size, without compromising the integrity of the monolayer. The Alhydrogel and Adju-Phos-mediated reorganization of phospholipid domains reported here supports the new mechanistic paradigm proposed by Shi and co-workers, and further suggests that lipid clustering is induced even in simple phospholipid membranes. The results present the basis for future exploration into lipid-mediated mechanisms of action for adjuvants.

Separation of liquid domains in model membranes induced with high hydrostatic pressure.

We have imaged the formation of membrane microdomains immediately after their induction using a novel technology platform coupling high hydrostatic pressure to fluorescence microscopy. After formation, the ordered domains are small and highly dynamic. This will enhance links between model lipid assemblies and dynamic processes in cellular membranes.

Cholesterol depletion using methyl-β-cyclodextrin.

Cholesterol is an essential component of mammalian cells. It is the major lipid constituent of the plasma membrane and is also abundant in most other organelle membranes. In the plasma membrane cholesterol plays critical physical roles in the maintenance of membrane fluidity and membrane permeability. It is also important for membrane trafficking, cell signalling, and lipid as well as protein sorting. Cholesterol is essential for the formation of liquid ordered domains in model membranes, which in cells are known as lipid nanodomains or lipid rafts. Cholesterol depletion is widely used to study the role of cholesterol in cellular processes and can be performed over days using inhibitors of its synthesis or acutely over minutes using chemical reagents. Acute cholesterol depletion by methyl-β-cyclodextrin (MBCD) is the most widely used method and here we describe how it should be performed to avoid the common side-effect cell death.

Phases and Fluctuations in Biological Membranes

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase separated domains in model membranes. However, this concept remains controversial due to the difficulty in directly observing these domains in intact cells. Here, we visualize ordered and disordered phase-like domains in intact B cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, establish that ordered domains are local environments that favor tyrosine phosphorylation, and present a minimal and predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling as well as a plausible mechanism of BCR activation via receptor clustering. More generally, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that may broadly impact signal transduction.

Abstract A229: Mechanism of action of perifosine on the mantle cell lymphoma line, Granta-519.

Abstract Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and sphingolipids and provides a scaffold for signal transduction. Altered raft assembly has been implicated in cancer progression. Alkylphospholipids have been used with promising specific cytotoxic effects in different types of cancer cells. These alkylphospholipids act by interaction with cell membranes and protein lipid rafts. Cell viability was determined by trypan blue assay. Cell cycle was evaluated by flow cytometry. Lipid rafts were isolated using sucrose density-gradient centrifugation after 12 hours of treatment with perifosine. Antibody microarray and western blotting was used to determine levels of key cellular proteins in special on cell signaling proteins. We showed that perifosine, an alkylphospholipid, targets raft-like domains in model membranes and induces apoptosis in mantle cell lymphomas (ED-50 20 μM - Granta-519) by activation of both extrinsic and intrinsic pathways. We observed an effect on the organization of the cell cycle, with increase of the population at G2/M phase (8,6% ± 0,6% to 27,4% ± 2,4%). Cyclin D1 decrease was detected at 24 hours of treatment. We also showed that perifosine downregulates NTAL/LAB (Non-T-cell activation linker/ Linker for activation of B-cells), an adaptor protein that is targeted to rafts by palmitoylation and specifically expressed in hematopoietic tissues. Moreover, perifosine induced a loss of NTAL/LAB in the lipid rafts of Granta-519 cells after 12 hours of treatment. Since NTAL/LAB may function as an adaptor protein in AKT signalization, we evaluate the effect of perifosine on the ATK pathway. We showed that perifosine lead to dephosphorylation of AKT and downstream components of AKT signaling. Moreover, in functional experiments, perifosine inhibited AKT activation by CD40L or IL-4 in Granta-519 cells, after few minutes of incubation, in a way similar to the specific PI3K inhibitor wortmannin. Treatment with methyl-β-cyclodextrin, a compound for cholesterol depletion, potentiated this effect. This suggests that NTAL/LAB translation is highly regulated and dependent on a functional AKT pathway. Our results indicate that perifosine acts on NTAL/LAB presumable by interference with its protein-lipid interactions and consequently AKT signaling. Our results demonstrate that a lipid raft targeting drug may present several effects on signal transduction, causing a severe toxicity to mantle cell lymphoma. Moreover, adaptor proteins like NTAL/LAB emerge as possible new therapeutic targets in lymphoma. This research was supported by FAPESP, CNPq and CAPES. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A229. Citation Format: Germano A. Ferreira, Carolina H. Thome, Guilherme A. dos Santos, Priscila S. Scheucher, Andreia M. Leopoldino, Ana M. Simão, Clarice Izume, Rodrigo A. Panepucci, Pietro Ciancaglini, Eduardo M. Rego, Vitor M. Faça, Lewis J. Greene. Mechanism of action of perifosine on the mantle cell lymphoma line, Granta-519. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A229.

Partitioning of liquid-ordered/liquid-disordered membrane microdomains induced by the fluidifying effect of 2-hydroxylated fatty acid derivatives

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses.

Formation of Raft-Like Assemblies within Clusters of Influenza Hemagglutinin Observed by MD Simulations

The association of hemagglutinin (HA) with lipid rafts in the plasma membrane is an important feature of the assembly process of influenza virus A. Lipid rafts are thought to be small, fluctuating patches of membrane enriched in saturated phospholipids, sphingolipids, cholesterol and certain types of protein. However, raft-associating transmembrane (TM) proteins generally partition into Ld domains in model membranes, which are enriched in unsaturated lipids and depleted in saturated lipids and cholesterol. The reason for this apparent disparity in behavior is unclear, but model membranes differ from the plasma membrane in a number of ways. In particular, the higher protein concentration in the plasma membrane may influence the partitioning of membrane proteins for rafts. To investigate the effect of high local protein concentration, we have conducted coarse-grained molecular dynamics (CG MD) simulations of HA clusters in domain-forming bilayers. During the simulations, we observed a continuous increase in the proportion of raft-type lipids (saturated phospholipids and cholesterol) within the area of membrane spanned by the protein cluster. Lateral diffusion of unsaturated lipids was significantly attenuated within the cluster, while saturated lipids were relatively unaffected. On this basis, we suggest a possible explanation for the change in lipid distribution, namely that steric crowding by the slow-diffusing proteins increases the chemical potential for unsaturated lipids within the cluster region. We therefore suggest that a local aggregation of HA can be sufficient to drive association of the protein with raft-type lipids. This may also represent a general mechanism for the targeting of TM proteins to rafts in the plasma membrane, which is of functional importance in a wide range of cellular processes.

Multimerizable HIV Gag derivative binds to the liquid-disordered phase in model membranes

During HIV assembly, a protein coat on the inner leaflet of the plasma membrane drives the formation of virus particles, and appears to induce the preferential accumulation of 'raft' lipids in the viral envelope, although the lipid raft concept mainly proposes microdomains of these lipids in the outer leaflet. The common hypothesis is that Gag preferentially associates with, and thereby probably induces, raft-like domains, because the protein is multimerized and specifically linked to two saturated acyl chains. To test this hypothesis, we constructed a minimal in vitro system in which we analysed the interaction of a Gag derivative, which could be triggered to multimerize, with a domain-forming model membrane resembling the inner leaflet of the plasma membrane. Confirming studies with authentic Gag, this Gag derivative only bound to membranes when it was multimerized, myristoylated and when phosphatidylinositol 4,5-bisphosphate was present in the membrane. Unexpectedly, however, the multimerized Gag derivative was largely excluded from ordered domains in model membranes. This suggests that the mechanism of membrane reorganization during HIV assembly does not simply result from a higher affinity of the clustered Gag membrane binding domain to ordered membrane domains, but involves more complex biophysical interactions or possibly also an additional protein machinery.

Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

Differential Dynamic and Structural Behavior of Lipid-Cholesterol Domains in Model Membranes

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo).

Identifying Membrane Lateral Organization by Contrast-Matched Small Angle Neutron Scattering.

Lipid domains in model membranes are routinely studied to provide insight into the physical interactions that drive raft formation in cellular membranes. Using small angle neutron scattering, contrast-matching techniques enable the detection of lipid domains ranging from tens to hundreds of nanometers which are not accessible to other techniques without the use of extrinsic probes. Here, we describe a probe-free experimental approach and model-free analysis to identify lipid domains in freely floating vesicles of ternary phase separating lipid mixtures.