New insights into the structure and hydration of a stratum corneum lipid model membrane by neutron diffraction

The structure and hydration of a stratum corneum (SC) lipid model membrane composed of N-(-hydroxyoctadecanoyl)-phytosphingosine (CER6)/cholesterol (Ch)/palmitic acid (PA)/cholesterol sulfate (ChS) were characterized by neutron diffraction. The neutron scattering length density across the SC lipid model membrane was calculated from measured diffraction peak intensities. The internal membrane structure and water distribution function across the bilayer were determined. The low hydration of the intermembrane space is a major feature of the SC lipid model membrane. The thickness of the water layer in the SC lipid model membrane is about 1 Å at full hydration. For the composition 55% CER6/25% Ch/15% PA/5% ChS, in a partly dehydrated state (60% humidity) and at 32°C, the lamellar repeat distance and the membrane thickness have the same value of 45.6 Å . The hydrophobic region of the membrane has a thickness of 31.2 Å . A decrease of the Ch content increases the membrane thickness. The water diffusion through the SC lipid model multilamellar membrane is a considerably slow process relative to that through phospholipid membranes. In excess water, the membrane hydration follows an exponential law with two characteristic times of 93 and 44 min. At 81°C and 97% humidity, the membrane separates into two phases with repeat distances of 45.8 and 40.5 Å . Possible conformations of CER6 molecules in the dry and hydrated multilayers are discussed.     

Arrangement of ceramide [EOS] in a stratum corneum lipid model matrix: new aspects revealed by neutron diffraction studies

The lipid matrix in stratum corneum (SC) plays a key role in the barrier function of the mammalian skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). Especially the unique-structured omega-acylceramide CER[EOS] is regarded to be essential for skin barrier properties by inducing the formation of a long-periodicity phase of 130 angstroms (LPP). In the present study, the arrangement of CER[EOS], either mixed with CER[AP] and CHOL or with CER[AP], CHOL and palmitic acid (PA), inside a SC lipid model membrane has been studied for the first time by neutron diffraction. For a mixed CER[EOS]/CER[AP]/CHOL membrane in a partly dehydrated state, the internal membrane nanostructure, i.e. the neutron scattering length density profile in the direction normal to the surface, was obtained by Fourier synthesis from the experimental diffraction patterns. The membrane repeat distance is equal to that of the formerly used SC lipid model system composed of CER[AP]/CHOL/PA/ChS. By comparing both the neutron scattering length density profiles, a possible arrangement of synthetic long-chain CER[EOS] molecules inside a SC lipid model matrix is suggested. The analysis of the internal membrane nanostructure implies that one CER[EOS] molecule penetrates from one membrane layer into an adjacent layer. A 130 angstroms periodicity phase could not be observed under experimental conditions, either in CER/CHOL mixtures or in CER/CHOL/FFA mixture. CER[EOS] can be arranged inside a phase with a repeat unit of 45.2 angstroms which is predominately formed by short-chain CER[AP] with distinct polarity.     

Localisation of partially deuterated cholesterol in quaternary SC lipid model membranes: a neutron diffraction study

This letter presents our first results in using the benefit of selective deuteration in neutron diffraction studies on stratum corneum (SC) lipid model systems. The SC represents the outermost layer of the mammalian skin and exhibits the main skin barrier. It is essential for studying drug penetration through the SC to know the internal structure and hydration behaviour on the molecular level. The SC intercellular matrix is mainly formed by ceramides (CER), cholesterol (CHOL) and long- chain free fatty acids (FFA). Among them, CHOL is the most abundant individual lipid, but a detailed knowledge about its localisation in the SC lipid matrix is still lacking. The structure of the quaternary SC lipid model membranes composed of either CER[AP]/CHOL-D6/palmitic acid (PA)/cholesterol sulphate (ChS) or CER[AP]/CHOL-D7/PA/ChS is characterized by neutron diffraction. Neutron diffraction patterns from the oriented samples are collected at the V1 diffractometer of the Hahn-Meitner-Institute, Berlin, measured at 32°C, 60% humidity and at different D₂O contents. The neutron scattering length density profile in the direction normal to the surface is restored by Fourier synthesis from the experimental diffraction patterns. The analysis of scattering length density profile is a suitable tool for investigating the internal structure of the SC lipid model membranes. The major finding is the experimental proof of the CHOL localisation in SC model membrane by deuterium labelling at prominent positions in the CHOL molecules.     

Effect of galactosylceramide on stratum corneum intercellular lipid synthesis in a three-dimensional cultured human epidermis model

Intercellular lipids in the stratum corneum (SC), such as ceramide (CER), free fatty acid (FFA), and cholesterol (CHOL), contribute to the formation of stable lamellar structures in the SC, making them important for skin barrier function. β-Galactosylceramide (GalCer) is a glycosphingolipid that is used in some cosmetics and quasi-drugs in anticipation of a moisturizing effect. GalCer promotes keratinocyte differentiation and increases CER production by increasing β-glucocerebrosidase (β-GCase) activity. However, few reports have described the mechanism of these effects, and detailed studies on the role of GalCer in intercellular lipid production in the SC have not been conducted. This study investigated the effect of GalCer on the metabolism and production of intercellular lipids in the SC in a three-dimensional cultured epidermis model. After reacting GalCer with a homogenate solution of three-dimensional cultured epidermis, GalCer was hardly metabolized. Treatment of the three-dimensional cultured epidermis with GalCer increased the expression of genes involved in the β-GCase metabolic pathway and promoted CER production. In addition, GalCer treatment reduced the expression of FFA metabolism-related genes as well as palmitic acid levels. In addition, transepidermal water loss, which is a barrier index, was reduced by GalCer treatment. These findings suggested that GalCer, which is hardly metabolized, affects the production of intercellular lipids in the SC and improves skin barrier function.     

The effect of the chain length distribution of free fatty acids on the mixing properties of stratum corneum model membranes

The stratum corneum (SC) plays a fundamental role in the barrier function of the skin. The SC consists of corneocytes embedded in a lipid matrix. The main lipid classes in the lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to examine the effect of the chain length of FFAs on the thermotropic phase behavior and mixing properties of SC lipids. Fourier transform infrared spectroscopy and Raman imaging spectroscopy were used to study the mixing properties using either protonated or deuterated FFAs. We selected SC model lipid mixtures containing only a single CER, CHOL and either a single FFA or a mixture of FFAs mimicking the FFA SC composition. The single CER consists of a sphingoid base with 18 carbon atoms and an acyl chain with a chain length of 24 carbon atoms. When using lignoceric acid (24 carbon atoms) or a mixture of FFAs, the CER and FFAs participated in mixed crystals, but hydration of the mixtures induced a slight phase separation between CER and FFA. The mixed crystalline structures did not phase separate during storage even up to a time period of 3 months. When using palmitic acid (16 carbon atoms), a slight phase separation was observed between FFA and CER. This phase separation was clearly enhanced during hydration and storage. In conclusion, the thermotropic phase behavior and the mixing properties of the SC lipid mixtures were shown to strongly depend on the chain length and chain length distribution of FFAs, while hydration enhanced the phase separation.     

Individual variation of human plantar stratum corneum lipids, determined by automated multiple development of high-performance thin-layer chromatography plates

The stratum corneum lipids are unique in composition and have been used frequently as a model system of the skin's lipid barrier. Automated multiple development (AMD) of high-performance thin-layer chromatography plates in combination with a 25-step gradient, based on methanol, diethyl ether and n-hexane separated the six major human plantar stratum corneum lipids. Post-chromatographic staining of these lipids with a solution of MnCl₂H₂SO₄ at 130°C or a solution of CuSO₄H₃PO₄ at 140°C allowed visualization of the lipids and quantification. The MnCl₂H₂SO₄ solution stained saturated fatty acids less intensity. Therefore, the CuSO₄H₃PO₄ solution was used for quantification and we found, on average, 2.06% (w/w) cholesterol 3-sulphate, 20.16% (w/w) free fatty acids, 20.25% (w/w) ceramides, 43.53% (w/w) non-esterified sterols, 4.56% (w/w) triacylglycerols and 9.4% (w/w) sterolesters in the human plantar stratum corneum extracts. The concentration of phospholipids was less than 1% (w/w). In addition, the lipid composition of twenty different human plantar stratum corneum extracts was determined. Statistics revealed a correlation between the ratio of free fatty acids and non-esterified sterols (r=0.832, p<0.01, n=20). Several control experiments proved that this correlation is not due to the extraction method, the post-chromatographic staining procedure or bacterial contamination of the stratum corneum.     

ATR-FTIR spectroscopy: a chemometric approach for studying the lipid organisation of the stratum corneum

The barrier function of skin resides in the lipid components of the stratum corneum, particularly their spatial organisation. FTIR spectroscopy has already been used as a relevant tool to study this lipid organisation: IR vibration band shifts have been attributed to the variations in lipid organisation induced by temperature. Our study included a stratum corneum model, composed of the three main lipids: palmitic acid as an example of fatty acids, cholesterol and ceramide III as an example of ceramide. Different films with various ratios of these lipids were studied. In our analytical strategy, the interest of using a chemometric analysis of global data obtained from ATR-FTIR spectra to highlight the main interactions involved in the molecular organisation of lipids has been demonstrated. Two kinds of interaction between the three main lipids have been shown: a non polar interaction between the long hydrocarbon chains and a polar interaction as the hydrogen bonding between polar functional groups. By varying the lipid ratio, we have shown first that the relative importance of each interaction was modified, second, that the induced modification of organisation can be detected by chemometric analysis of the ATR-FTIR spectra. The role of each kind of lipid in the organisation has been discussed. In conclusion, associating the ATR-FTIR with chemometric treatment is a promising tool: firstly, to understand the consequence of lipid relative compositions on the structural organisation of the stratum corneum, secondly, to show the relationship between lipid organisation and percutaneous penetration data. Indeed, this methodology will be transposed to in vivo studies with IR measurements through a probe.     

Physical and chemical perturbations of the supramolecular organization of the stratum corneum lipids: In vitro to ex vivo study

Background: FTIR spectroscopy is classically used to study the supramolecular organization of the stratum corneum lipids. Exposure to UV_A is responsible for a small decrease in packing observed on cutaneous lipid films. Methods: Lipid films and human skin biopsies were either exposed to UV_A irradiation of 120 J/cm², UV_B irradiation of 0.15 J/cm² or put in contact with ethanol. Using FTIR in vitro and IR microspectroscopy ex vivo provided information on: i) the precise localisation of the stratum corneum in the skin, ii) its thickness, and iii) the organization of its constituted lipids. Results: Different action modes were observed for UV irradiation and the contact with ethanol with a certain destabilisation of the lipidic layer. Ethanol was also found to be responsible for the creation of pores. The destabilisation of the lipid cement was mainly observed ex vivo. Conclusion: The barrier function of the skin is affected by the action of physical and chemical external agents at the molecular level. The increased laxity of the lipid packing could enable the percutaneous penetration velocity of actives.     

Investigation of stratum corneum lipid model membranes with free fatty acid composition by neutron diffraction

The outermost epidermal layer, the stratum corneum (SC), is the main skin barrier. Studies of SC model systems enable characterization of the influence of individual lipids on the organization of the SC lipid matrix, which is the main pathway of water through the skin. This work presents a neutron diffraction study of the SC model membranes based on short-chain ceramide 6 with nearly realistic composition of free fatty acids (FFA) at physiological temperature of the SC. The influence of FFA and the effect of cholesterol–cholesterol sulfate substitution on the structure and hydration of the SC model membranes are described. The structure of the SC membrane with FFA is close to the structure of the earlier studied SC membrane based on short-chain palmitic acid (PA) and does not vary significantly under changes of the ratio of the main membrane components. FFA accelerates membrane swelling at the same low level of hydration of both PA- and FFA-containing membranes. The substitution of cholesterol sulfate by cholesterol in the membrane composition decreases membrane swelling and leads to phase separation in the model system.     

An electron microscopic study of non-fixed and non-dehydrated normal human stratum corneum

Summary This is an electron microscopic study of non-fixed and non-dehydrated normal human stratum corneum from the lumbar region.Non-stained sections have a low contrast. In sections examined 3 days after skin biopsy the cytoplasm of the cells shows a uniform contrast or exhibits dark and light areas. A single layer delimits the cytoplasm from the intercellular space. The latter is partly filled out with substance.In sections stained 2 to 4 days after skin biopsy the fibrils are distinct. On the basis of the variations in their opacity and ultrastructure three types of horny cells are clearly distinguishable. In cells of type 1 intensely stained keratohyalin and less opaque fibrillar substance occur. A distinct keratin pattern is not found. In cells of type 2 the fibrils show areas with distinct kerytohyalin and keratin pattern and transitional phases between these two stages of fibrillar differentiation. The keratin pattern representing the final stage of the fibrillar differentiation process is visualized through a successive discoloration of the filaments, whereas the interfilamentous substance retains the opacity of the keratohyalin. In cells of type 3 the entire fibrillar substance exhibits a keratin pattern. This consists of less opaque filaments with a diameter of 74 Å. The septa representing the interfilamentous substance are estimated as 30 Å at their thinnest points. These observations of the fibrils are completely comparable to the findings in fixed and dehydrated normal human stratum corneum.In sections stained particularly more than 18 days after skin biopsy the fibrils exhibit pronounced changes in their staining properties with concomitant decrease in distinctness or a complete extinction of the keratin pattern.The observations of the modified plasma membrane and the intercellular space in stained sections correspond to the findings in fixed and dehydrated normal human stratum corneum. The modified plasma membrane and the structures in the intercellular space appear with equal distinctness, whether the sections are stained 2 to 4, 6 to 12 or 14 to 21 days after skin biopsy.This investigation was supported by grants from the Edvard Welander Foundation and from the Swedish Medical Research Council (B71-12X-2708-03).     

Two new methods for preparing a unique stratum corneum substitute

Stratum corneum lipids play an important role in the barrier function of the skin. An in vitro permeation model consisting of synthetic lipids has previously been developed to replace human stratum corneum (SC) in permeation studies. This model is referred to as the stratum corneum substitute (SCS). In order to improve its reproducibility and to increase the efficiency in preparing the SCS, two new preparation methods are developed. Subsequently the properties of the SCS prepared by the various methods, i.e. the manual airbrush method, the rotor airbrush method and the linomat method, are investigated. The results show that the SCS prepared with the various methods share the properties of a uniform lipid composition and lipid distribution. Furthermore, irrespective of the preparation method, the lipids form crystalline lamellar phases, mimicking the lipid organization and orientation in human SC. As a result, permeation profiles of benzoic acid through SCS are very similar to human SC. The rotor method increases the efficiency and reproducibility of the manual airbrush method, while the linomat method reduces the lipid loss during preparation and results in SCS with a more uniform membrane thickness. In conclusion, the linomat method was chosen as the preferred method for preparing the substitute.     

A network model of successive partitioning-limited solute diffusion through the stratum corneum

As the most exposed point of contact with the external environment, the skin is an important barrier to many chemical exposures, including medications, potentially toxic chemicals and cosmetics. Traditional dermal absorption models treat the stratum corneum lipids as a homogenous medium through which solutes diffuse according to Fick's first law of diffusion. This approach does not explain non-linear absorption and irregular distribution patterns within the stratum corneum lipids as observed in experimental data. A network model, based on successive partitioning-limited solute diffusion through the stratum corneum, where the lipid structure is represented by a large, sparse, and regular network where nodes have variable characteristics, offers an alternative, efficient, and flexible approach to dermal absorption modeling that simulates non-linear absorption data patterns. Four model versions are presented: two linear models, which have unlimited node capacities, and two non-linear models, which have limited node capacities. The non-linear model outputs produce absorption to dose relationships that can be best characterized quantitatively by using power equations, similar to the equations used to describe non-linear experimental data.     

Combined LC/MS-platform for analysis of all major stratum corneum lipids,and the profiling of skin substitutes

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10 μl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, ‘high-throughput’ profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved.     

Effect of dimethyl sulfoxide on the phase behavior of model stratum corneum lipid mixtures

Dimethyl sulfoxide (DMSO), an efficient transdermal enhancer, is proposed to alter the skin barrier by, at least partially, disturbing the lipid phase of the stratum corneum (SC). We have investigated, using differential scanning calorimetry and vibrational microspectroscopy, the effect of DMSO on the phase behavior of a lipid mixture formed by N-palmitoyl-d-erythro-sphingosine, deuterated palmitic acid, and cholesterol, mimicking the SC lipid phase. Our results reveal that DMSO favors the disordering of the lipid acyl chains. Moreover, the effect of DMSO is strongly concentration dependent and this dependence is reminiscent of that describing the DMSO transdermal enhancement. DMSO-induced fluidification affects primarily the fatty acid in the mixture. Therefore, it is proposed that the molecular mechanism of the transdermal transport enhancement caused by DMSO is associated with its H-bonding properties; its presence alters the interfacial H-bond network involving the fatty acid molecules and consequently the cohesive lipid packing.     

Swelling of intercellular lipid lamellar structure with short repeat distance in hairless mouse stratum corneum as studied by X-ray diffraction

Lamellar structures of intercellular lipids in stratum corneum of hairless mouse were studied at various water contents by small-angle X-ray diffraction. At room temperature there are at least two lamellar structures, long and short lamellar structures, with repeat distances of 13.6 and around 6 nm, respectively. The long lamellar spacing is almost constant over the water content from 0% w/w to 80% w/w that is consistent with the previously reported results. For the short lamellar structure we found that with increasing the water content the lamellar spacing becomes larger, that is, from 12 to 50% w/w the short lamellar spacing increases from 5.8 to 6.6 nm. In addition to the previously reported result that at the water content of about 20% w/w the X-ray diffraction peak for the long lamellar structure becomes sharp, we found that this is also the case for the short lamellar structure. Below the water content of about 12% w/w the X-ray diffraction peak for the short lamellar structure dies out and conversely above the water content of about 50% w/w it becomes weak and finally merges into the second-order diffraction peak for the long lamellar structure. Considering the matching of the long lamellar spacing that is unchanged with the water content and twice the short lamellar spacing that changes as a function of the water content, it is likely that the swelling of the short lamellar structure plays an important role in the regulation of water stored in stratum corneum.     

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.     

Evidence of free fatty acid interdigitation in stratum corneum model membranes based on ceramide [AP] by deuterium labelling

This research paper provides direct evidence concerning the localisation of free fatty acids in stratum corneum lipid model membranes. We employed partially deuterated free fatty acids to gain further information about the assembly of a stratum corneum lipid model membrane based on a ceramide of the phytosphingosine-type (ceramide [AP]) with particular respect to the position of the deuterated groups of the free fatty acids. The application of behenic-22,22,22-d₃-acid and cerotic-12,12,13,13-d₄-acid confirmed that the short-chain ceramide [AP] forces the longer-chained free fatty acids to incorporate into the bilayer created by ceramide [AP]. The ceramide [AP] molecules determine the structural assembly of this model membrane and obligate the long-chain free fatty acids to either arrange inside this formation or to separate as a fatty acid rich phase.     

Hydration of POPC bilayers studied by 1H-PFG-MAS-NOESY and neutron diffraction

The stability of lipid bilayers is ultimately linked to the hydrophobic effect and the properties of water of hydration. Magic angle spinning (MAS) nuclear Overhauser enhancement spectroscopy (NOESY) with application of pulsed magnetic field gradients (PFG) was used to study the interaction of water with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers in the fluid phase. NOESY cross-relaxation between water and polar groups of lipids, but also with methylene resonances of hydrophobic hydrocarbon chains, has been observed previously. This observation led to speculations that substantial amounts of water may reside in the hydrophobic core of bilayers. Here, the results of a quantitative analysis of cross-relaxation in a lipid 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC)/water mixture are reported. Coherences were selected via application of pulsed magnetic field gradients. This technique shortens acquisition times of NOESY spectra to 20 min and reduces t ₁-spectral noise, enabling detection of weak crosspeaks, like those between water and lipids, with higher precision than with non-gradient NOESY methods. The analysis showed that water molecules interact almost exclusively with sites of the lipid–water interface, including choline, phosphate, glycerol, and carbonyl groups. The lifetime of lipid–water associations is rather short, on the order of 100 ps, at least one order of magnitude shorter than the lifetime of lipid–lipid associations. The distribution of water molecules over the lipid bilayer was measured at identical water content by neutron diffraction. Water molecules penetrate deep into the interfacial region of bilayers but water concentration in the hydrophobic core is below the detection limit of one water molecule per lipid, in excellent agreement with the cross-relaxation data. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.     

Influence of chemical and freezing fixation methods in the freeze-fracture of stratum corneum

A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum-carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist.