Imaging Ellipsometry of Spin-Coated Membranes: Mapping of

Jan 2, 2013 - Jes Dreier , Jonathan Brewer , and Adam Cohen Simonsen ... Deirdre A. Costello , Chih-Yun Hsia , Jean K. Millet , Teresa Porri , and Sus...
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Imaging Ellipsometry of Spin-Coated Membranes: Mapping of Multilamellar Films, Hydrated Membranes, and Fluid Domains Mette Marie Bruun Nielsen and Adam Cohen Simonsen* MEMPHYS - Center for Biomembrane Physics Department of Physics, Chemistry and Pharmacy University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark ABSTRACT: Imaging ellipsometry (IE) has been applied to generate laterally resolved thickness maps of spin-coated membranes in both the dry and fully hydrated state. Spin-coating offers a convenient preparation method for stacked supported membranes, and in-depth thickness maps for such films can be measured by IE, thereby going beyond topography measurements of the top surface. We find that dry lipid films of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) have a highly ordered multilamellar structure which allows counting of the number of individual bilayers in a thick film from the progression in a concentration series. The average film thickness is approximately proportional to the coating concentration with a constant of proportionality of 5.2 nm/mM and 6.2 nm/mM for POPC and DSPC (1,2-distearoyl-sn- glycero-3-phosphocholine), respectively. The root-mean-square roughness of the dry films is also approximately proportional to concentration with constants of 3.7 nm/mM (DSPC) and 0.87 nm/mM (POPC). Fully hydrated POPC membranes with several stacked bilayers show decreasing thickness for increasing temperature. An apparent excess in thickness by 1.2 nm for the proximal membrane can possibly be linked to the presence of a structured water film next to the solid support. This is supported by modeling of spectroscopic data. Thickness maps of double supported ternary membranes show resolvable liquid-ordered domains in the second membrane while domains are below the resolution limit in the proximal membrane. A thickness difference of 1.69 and 1.89 nm between the liquid-ordered (lo) and liquid-disordered (ld) phases is found for two different ternary membrane compositions. This is approximately twice the height difference measured by AFM on domains, thus indicating that the relative excess thickness of the lo phase is symmetrically distributed. copy (AFM), fluorescence microscopy, quartz crystal microbalance (QCM), and scattering techniques. The techniques often provide complementary information, and each technique carries a unique set of measurable physical quantities. AFM measures the surface topography with a maximum image size around 100 × 100 μm 2, a typical lateral resolution of 1−2 nm, and a vertical resolution below 0.1 nm. Epifluorescence microscopy maps the spatial distribution of fluorescent markers with a typical lateral resolution of 0.5 μm and image sizes in the micrometer to millimeter range. Most scattering techniques are averaged over large sample regions (mm to cm) but provide detailed structural information on short length scales. As an example, grazing incidence X-ray diffraction (GIXD) can reveal the positional ordering of membranes in the gel phase.8 Many characterization techniques are invasive in the sense that they impose special conditions such as sample drying, mechanical contact to the sample, or addition of labeling molecules. Ellipsometry9,10 has gradually emerged as a highly useful technique for characterizing soft interfaces11,12 and membranes due to its noninvasive nature and the potential for obtaining accurate thickness information in stratified systems under

1. INTRODUCTION Lipid bilayers constitute the structural backbone of biomembranes, and simple lipid bilayers can exist in several well-defined thermodynamic phase states. In contrast, cellular biomembranes are highly complex soft interfaces responsible for the confinement of cells and organelles. To reveal the underlying physical principles governing biomembrane structure and function, model membranes with simple compositions have become indispensable experimental tools. An example is the importance of the liquid-ordered (lo) phase which is a thermodynamic phase regulated by cholesterol1,2 and which forms the physical basis for the phenomenological raft model in cell biology.3 Fluid domains in the lo phase can be studied in ternary membranes which have today become the most widely adopted equilibrium model for raft phenomena.4,5 The development of model membrane studies is mainly taking place in two areas: (1) Sample preparation and sample design and (2) invention and improvement of characterization tools. Supported membranes are some of the most widely used models with a particular advantage being the wide range of applicable characterization tools6 and the planar geometry which simplifies image analysis. Supported membranes have been realized for a wide range of systems and support materials as reviewed recently.7 Among the techniques that are used for characterizing supported membranes are atomic force micros© 2013 American Chemical Society

Received: November 23, 2012 Revised: December 28, 2012 Published: January 2, 2013 1525

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Sphingomyelin (Brain, Porcine) (SM), and cholesterol, all purchased from Avanti Polar Lipids (Alabaster, AL). All lipids were dissolved in a solvent composed of (97:3) hexane and methanol. All solvents and chemicals (Sigma-Aldrich) were HPLC grade quality, and ultrapure Milli-Q water (18.3 MΩ·cm) was used in all steps involving water. For results on hydrated membranes, a 150 mM NaCl solution was used as the hydration medium. 2.2. Sample Preparation. The substrate of the supported membranes were Si(100) wafers (Semiconductor Wafer Inc.) cut into 15 × 15 mm2 or 15 × 25 mm2 pieces for experiments in air and water, respectively. The wafers were cleaned in two steps: Immersion in a hot (80 °C) solution of (1:1:4) 30% NH3(aq), 35% H2O2, and H2O followed by rinsing in water and drying. Immediately prior to spin-coating, wafers were plasma-cleaned (PDC-002, Harrick Plasma, Ithaca, NY) in atmospheric air plasma (300 mTorr) for 20 min to remove traces of organic residues. The thickness of the SiO2 oxide layer was measured independently for a number of substrates and was always found to be close to 2.0 nm with maximum deviations of ±0.2 nm. We have used a fixed oxide thickness of 2.0 nm for all maps in this work. We have tested (before/after) that our plasma treatment is not measurably affecting the thickness of the oxide film, nor does it change the sample in other ways as measured by ellipsometry. Dry spin-coated lipid films were prepared using a lipid stock solution in hexane−methanol (97:3). A volume of 80 μL of the lipid stock was applied to the silicon substrate and spun on a Chemat Technology, KW-4A spin-coater (Chemat Technology Inc.) at 3000 rpm for 40 s. The sample was then placed under vacuum until use to maximize evaporation of solvents. The spin-coated films were either measured directly in the dry state or hydrated in the ellipsometry fluid cell (Acccurion). For the hydrated samples, buffer was added to the fluid cell containing the spin-coated lipid film, and the immersed sample was heated to 55 °C for minimum 1 h. For complete hydration, the temperature must be maintained above the main phase transition temperature for the membrane under study. While at elevated temperature (55 °C) the sample was rinsed by a vertical flow of buffer toward a spot on the center of the sample. This was performed with a manual Finnpipette (Thermo Scientific) set to 300−500 μL, operated in vertical orientation and with the tip held at 1−2 mm from the sample surface. Washing releases membrane material from the surface which was removed from solution by purging the liquid cell >10 times with pure buffer. The flushed spot on the sample (1−3 mm diameter) contains a single membrane, while regions with multiple membranes are located outside this spot. For further details, see refs 17 and 18. 2.3. Imaging Ellipsometry and Modeling. Ellipsometry measurements were performed using an EP3se spectroscopic imaging ellipsometer (Accurion, GmbH, Göttingen, Germany) under ambient conditions at room temperature. The instrument is operated as a standard null-ellipsometer with the following components in the light path: polarizer, compensator, sample, objective, analyzer, and camera, i.e., a setup equivalent to a conventional PCSA-ellipsometer but using focusing and a camera for detection of the ellipsometry signal. The setup enables measurement and lateral mapping of the ellipsometry angles, Δ and Ψ, describing the ratio of the reflection coefficients across the sample surface: tan(Ψ)exp(iΔ) = rp/rs, with rp and rs being the Fresnel reflection coefficients for the light of p and s polarization, respectively. Overall focused images under oblique conditions are obtained with focus scanning and subsequent image reconstruction. Regions of interest (ROIs) on the sample can be selected for local ellipsometry measurements averaged over all pixels in the ROI. Maps of Δ and Ψ are created by recording a sequence of images for varying filter (polarizer and analyzer, respectively) positions and subsequently determining the null in each pixel. For ROI measurements we used the fixed compensator azimuth nulling scheme with four-zone nulling which provides more accurate values of Δ and Ψ than one-zone measurements. The light source is a 658 nm laser (used for mapping) or a xenon lamp with filter monochromator providing 44 wavelengths (bandwidths of ±6 to ±20 nm) in the wavelength range 363.7 to 1001.7 nm

natural conditions. In recent years imaging ellipsometry (IE) has been demonstrated as a powerful technique for mapping of the ellipsometry signal and through subsequent modeling to obtain thickness maps of soft interfaces such as membranes.13−16 IE is typically performed as an extension of classical null-ellipsometry where the ellipsometry angles Δ and Ψ are mapped with a camera sensor over a small lateral region of the sample. This capability is unique in the sense that ultrasoft and fragile biomolecular interfaces can be morphologically characterized. This opens the potential for thickness mapping of membrane domains and biomolecular binding events in complex model systems. IE does not require labeling of the sample, is noninvasive, and can be performed in native aqueous conditions. The lateral resolution of IE is typically 1−2 μm while the thickness resolution depends on the contrast in refractive index between the layers of the sample but can go down to around 0.1 nm for optimal samples. The substrate for IE is, for most applications, limited to silicon wafers. Transparent materials such as mica, although an excellent substrate for supported membranes, result in ellipsometry data that are difficult to analyze, due to subsurface reflections and loss of lateral resolution. Reproducible fabrication of high-quality supported membranes with a general lipid composition is essential for model studies. Classical vesicle fusion and Langmuir−Blodgett are frequently used but often have to be tested and optimized for specific system compositions. Moreover, fabrication of stacked supported membranes is desirable for studying membrane− membrane interactions and for decoupling the topmost membranes from the solid support. However, stacked membranes are difficult to prepare with the methods mentioned above. When using spin-coating as a method for preparing supported membranes,17,18 the dry lipids typically organize in multilamellar films whose thickness depends on the coating concentration.19,20 The dry lipid film forms the precursor for making a hydrated supported membrane. The method is much less sensitive to the lipid composition than classical fabrication methods because deposition on the support takes place from an organic solvent which governs wetting of the substrate and the coating formation. Spin-coated lipid films are not only interesting as precursors for hydrated membranes but are generally useful biomolecular coatings with potential use in sensors or for improving the biocompatibility of material surfaces. For these applications, a better understanding of the dry lipid film is necessary.21 In this study we apply IE to characterize lipid films in dry and hydrated states. Thickness maps are created, which reveal how the multilamellar thickness profile develops as the lipid concentration is increased. We demonstrate thickness maps for fully hydrated multilamellar membranes showing thickness variations between individual membranes in the stack. Finally, we demonstrate IE thickness maps of liquid-ordered/liquiddisordered domain coexistence in the second membrane of a double membrane system. These data are complementary to AFM data of fluid domains in the sense that the full membrane thickness is measured rather than only the surface topology.

2. MATERIALS AND METHODS 2.1. Materials. The lipids used in this study are 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn- glycero-3-phospho choline (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phophocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoscholine (DOPC), 1526

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Figure 1. Principles of the map analysis. (A, B) Maps of the ellipsometry angles Δ and Ψ for a dry POPC film spin-coated at a concentration of 5 mM. The gray scales show the range of angles or thicknesses in the maps. The Δ map contains adequate contrast for analysis while the signal-tonoise ratio is weaker in the Ψ image. (C) Thickness map in nm obtained from an interpolation between the Δ map values and the lipid thickness in a slab model of the sample. A 3D-rendition of the thickness map is displayed in panel D. A schematic illustration of the lipid structure of dry (E) and hydrated (F) lipid films and the slab models (G) used for modeling and construction of thickness maps. For hydrated samples, air is replaced by H2O in the model. All scalebars are 20 μm .

Figure 2. Series of thickness maps for dry POPC films coated at varying lipid concentrations. The concentrations used were as follows: 0.25 mM (A), 1 mM (B), 2 mM (C), 3 mM (D), 4 mM (E), 5 mM (F). The gray scales show the thickness range for each map in nanometers. (G) Histograms of the thicknesses in images A−F. All histograms are normalized to a range of 1.0 and vertically displaced for clarity. The vertical gridlines in panel G indicate thickness of the first monolayer and of the subsequent bilayers. All scalebars are 20 μm . The frequency-dependent complex refractive index n(ω) + ik(ω) of the dry and hydrated lipid films are input to the modeling of the experimental ellipsometry data. In a rigorous treatment, the anisotropy of the lamellar lipid film as well as membrane phase state, acyl chain length, and temperature would have to be taken into account. Optical anisotropy of lipid monolayers23,24 and bilayers25 have been characterized, resulting for uniaxial films in two refractive indices n⊥ and n∥. We use an isotropic average n2av = 1/3(n2⊥ + 2n2∥) due to the restrictions of null ellipsometry in treating anisotropic films. Given the scatter in literature values of the anisotropic refractive index for lipid films and the intrinsic uncertainties in our experimental data, we use a single fixed value of nav = 1.50 and k = 0 for all thickness maps in this paper. This procedure is in line with previous work on supported membranes.15

for spectroscopic ellipsometry of selected ROIs. The reflected light is collected with a 20× (NA = 0.35) or a 50× (NA = 0.45) objective, both with a lateral resolution of approximately 1 μm. For experiments in air, the angle of incidence (AOI) was fixed at 42°, while for measurements of hydrated samples in the fluid cell, the AOI is fixed at 60°. The fluid cell is temperature controlled using a Peltier device with water cooling (Jumo IMAGO 500 controller). Analysis and modeling of ellipsometry data were performed with the software EP4Model (1.0.1) (Accurion). The software performs modeling of the ellipsometry angles (Ψ, Δ) by calculating Fresnel coefficients for a slab model of the sample using tabulated or measured data for the optical properties of the slab layers. For the creation of thickness maps, all parameters in the slab model are fixed except for the thickness d of the lipid film. The thickness map is created in one of two ways: (1) By simple interpolation between d and Δ in each pixel (fast) or (2) by fitting Δ to the model in each pixel (slower). By carefully testing both procedures on our samples, we found that dry lipid films could be accurately mapped using the interpolation method whereas for hydrated samples the single pixel fitting was necessary. The optical functions of crystalline Si are well-known and implemented in the EP4Model simulation software,22 and the thickness of the substrate is considered semi-infinite in the model calculations. As for the SiO2 overlayer, we use the tabulated SiO2 optical functions implemented in the EP4Model and determine the SiO2 layer thickness by fitting to spectroscopic data (not shown).

3. RESULTS AND DISCUSSION Spin-coated lipid films are known to have a highly organized multilamellar structure when characterized topographically with AFM.17,21 To further validate the structure, ellipsometry maps can provide the thickness profile of this layered film. Figure 1A and 1B shows a typical example of the appearance of Δ and Ψ maps for a dry film (POPC). A significant aspect to notice in Figure 1 is the fact that the Δ angles vary over a range of 10° whereas Ψ varies over only 1.5° which is close to the noise level. The reason for this difference 1527

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Figure 3. Series of thickness maps for dry DSPC films coated at varying lipid concentrations. The concentrations used were: 0.25 mM (A), 1 mM (B), 2 mM (C), 3 mM (D), 4 mM (E), 5 mM (F). The gray scales show the thickness range for each map in nanometers. (G) All histograms are normalized to a range of 1.0 and vertically displaced for clarity. All scalebars are 20 μm.

is that the change in the wave phase (contained in Δ) upon reflection from this thin film is stronger than the amplitude modulation (contained in Ψ). For this reason, we calculate the thickness map (Figure 1C) from a model fit to the Δ map only. This is also the case for all subsequent thickness maps in this work which are based only on the Δ maps. The thickness map in Figure 1C clearly reveals the lamellar structure of the lipid film with distinct areas corresponding to different numbers of stacked bilayers. The 3D rendition in Figure 1D shows the thickness map reconstructed as a 3D surface. Because the Si(100) substrate has a uniformly flat topography, our in-depth thickness map may be translated meaningfully to a topography map of the lipid film surface. For films with a nonplanar substrate, the translation from film thickness to topography may be less straightforward. The conformation of the lipid coating in dry and hydrated conditions is shown in Figure 1E,F. These structural models for the lipid films have been validated and discussed previously.17,18,21 Briefly, the dry lipid film, as created during spincoating from the solvent phase, consists of a stack of inverted bilayers with the lipid acyl chains facing the ambient air and the headgroups facing the solid substrate. Upon sample hydration, a stack of hydrated lipid bilayers will form, where the number of bilayers can be controlled by inducing liquid flow toward the sample surface. In particular, single and double supported membranes can be prepared with this method, and these are highly useful for model membrane studies. The slab models of the dry and hydrated samples used for analyzing the ellipsometry maps are shown in Figure 1G. Thickness maps for a concentration series of dry POPC films is shown in Figure 2A−F. These samples were created using lipid solutions with concentrations in the range of 0.25−5 mM. The layered sample structure is clearly visible in Figure 2C,E,F and less so in the other thickness maps. As discussed previously,17 the individual bilayers of a spin-coated lipid film exhibit characteristic dewetting patterns with holes/patches created as the film breaks up during solvent evaporation in the spin-coating process. The characteristic length scale of the dewetting pattern has been found to vary among samples and for different regions on the same sample. If the dewetting pattern is smaller than the lateral resolution limit of the imaging ellipsometer (∼2 μm), then individual layers cannot be discerned. This is the case in Figure 2A,B,D, where intermediate thickness are recorded (see below).

It is helpful to convert the thickness maps into histograms representing the distribution of thicknesses for all pixels in a given map. This is shown in Figure 2G for the maps in Figure 2A−F. Similar histograms are often obtained from AFM topography images, but in the case of AFM, the height distribution is frequently distorted or inaccurate due to the various nonlinear background subtraction procedures that are unavoidable in most AFM images. In contrast, the thickness histograms created from ellipsometry maps are free from background artifacts and can reveal distinct layered features of the sample, as shown below. The sequence of histograms in Figure 2G reveal a systematic increasing trend in thickness for increasing coating concentrations, where the film grows by increasing the average number of bilayers in the films. Overlapping peaks for subsequent samples allow us to determine the number of bilayers in each sample region. As an example, the tallest (brightest) regions of Figure 2F correspond to five bilayers and one monolayer. The vertical dotted lines in Figure 2G indicate the approximate thickness of the first monolayer and of the subsequent bilayers. Note that the histograms of the wellresolved samples (C, E, F) have peaks on the grid lines, while those of other samples (B, D) have thickness peaks at intermediate values. This is a result of the layers not being fully resolved laterally in these cases. The monolayer thickness in panel A is lower than half a bilayer thickness but corresponds well to recent AFM data for DOPC.21 Strong lipid−substrate interactions are likely to influence the structure of the dry proximal monolayer and reducing its thickness. Thickness maps for a concentration series of the saturated lipid DSPC are shown in Figure 3A−F as a comparison with POPC. The thickness pattern is distinctly different from POPC, which indicates that the lipid chemistry including the chain melting temperature is important for the film structure. Upon spin-coating, a complex self-organization process takes place during film thinning and solvent evaporation. Changes in film viscosity, lipid diffusivity, and substrate−lipid interactions during this process are important factors that influence the final film structure. When considering the thickness histograms for DSPC, we again observe a single monolayer for the lowest coating concentration of 0.25 mM. The next concentration of 1 mM produces a total thickness around 10 nm corresponding to a monolayer and a bilayer. For the subsequent higher concentrations, the peak around 10−12 nm is preserved 1528

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commonly employed in roughness analysis: Rq = (1/N∑i N= 1(di − dav)2)1/2. Comparing the roughness values of DSPC films with the images of Figure 3, it is clear that the larger roughness values originate from the formation of thick multilayered patches (Figure 3C−F) in contrast to POPC which appears to maintain a more uniform layered film structure at high coating concentrations. The dry spin-coated lipid films function as precursor structures for the formation of supported membranes by hydration. Following hydration in saline solution (150 mM NaCl) and flushing of the sample surface, we measure IE thickness maps of the hydrated POPC film at two temperatures, as shown in Figure 5A (55 °C) and 5B (20 °C). In general, the

while broader features extending up to 60 nm appear. This indicates that a complete monolayer and bilayer is initially formed and that subsequently patches of much thicker stringlike multilayers are formed as evident in Figure 3C−F. Most likely, the nanoscale structure of the patches is still ordered and lamellar but with lateral dimensions below the resolution limit of the IE technique. The reason for the formation of these heterogeneous features in DSPC films can be differences in viscosity or wetting properties during solvent evaporation which promotes breakup of the DSPC film. The POPC and DSPC film growth can conveniently be characterized in terms of the global film thickness obtained as an average over the thicknesses in the pixels of an image. Figure 4A shows the mean lipid thickness dlipid plottet versus the

Figure 5. Thickness maps of a system of stacked and fully hydrated POPC membranes at temperatures of 55 °C (A) and 20 °C (B). Greyscales show the thickness range for each map. Thickness histograms in panel C were constructed from the maps in panels A and B. Histograms are normalized to a range of 1.0 and vertically displaced. Symbols I, II, and III indicate the location and thickness of membranes nos. 1, 2, and 3 as counted from the solid substrate. The peak positions were determined by fitting Gaussian functions to each peak of the histogram. All scalebars are 20 μm .

Figure 4. Concentration plots of the mean (A) and standard deviation (B) of the film thickness generated from thickness maps. Data displayed for dry POPC and DSPC films. Insert in panel A shows the line slope which is a useful parameter for predicting film thickness based on the coating concentration.

coating concentration ccoat. For both lipids, we find approximately a proportional relationship: dlipid = αlipidccoat, in agreement with previous findings from X-ray diffraction19,20 and semiempirical modeling.26 We determine the constant of proportionality (coating constant) to be αPOPC = 5.3 nm/mM and αDSPC = 6.2 nm/mM. The fact that the coating constant is comparable for two quite different lipids indicates that the total thickness deposited by spin-coating is quite robust against changes in the lipid composition. The standard deviation σlipid of the thickness distribution is shown in Figure 4B and shows generally much higher values for the DSPC film as compared to POPC. The value of σDSPC also increases faster than σPOPC with respect to ccoat. A linear fit of the type σ = βccoat yields the constants βDSPC = 3.7 nm/mM and βPOPC = 0.87 nm/mM. Because our Si substrate is flat, the standard deviation of the film thickness could be interpreted as a surface roughness parameter. The standard deviation is in fact, formally identical to the root-mean-square-roughness Rq

sample surface for the hydrated lipid films contains multiple stacked bilayers with the number of bilayers varying with the lateral position on the sample. This is in agreement with our previous results on spin-coated membranes using fluorescence microscopy.27 For both sample regions in Figure 5, the images reveal the presence of three distinct film thicknesses indicated as I, II, and III. On the basis of the thickness values, we interpret these as corresponding to regions with one, two, and three stacked bilayers. The thickness histograms constructed from the thickness maps are shown in Figure 5C. For both temperatures, the histograms exhibit three nearly Gaussian peaks originating from regions I, II, and III in the thickness maps. With this information we can extract useful information on the thickness of the individual layers. By Gaussian fitting to the peaks of the histogram, we identify the peak positions as indicated in Figure 5C. The peak-to-peak intervals provide the thicknesses of the 1529

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Figure 6. Spectroscopic analysis examining the possibility of an adjoining water film on the Si(100) substrate. The sample is composed of a hydrated, stacked POPC membrane measured at 20 °C. The ellipsometry contrast image in panel A indicates two regions of interest (Roi0, Roi1) which have been measured spectroscopically. The lipid structure of these regions is indicated in (B). Spectroscopic Δ-fits (model inserted) for the lipid thickness d in Roi0 and Roi1 (C) confirms that the thicknesses for two regions correspond to double and single membranes, respectively. The proximal membrane has an excess thickness of 1.2 nm compared to the secondary membrane. Attempts to model the excess thickness by a bulk (n = 1.33) H2O layer are unsuccessful for all layer thicknesses, as shown in panel D. Attempts to model the excess thickness with a dielectric H2O layer with increased n is, however, successful as shown in panel E. In the last case, the layer thickness and the refractive index are strongly coupled and several equivalent fits can be obtained.

same slab model as in Figure 1G confirms that the proximal membrane has an excess thickness compared to the second membrane. To test if the excess thickness can be modeled by a dielectric water layer, we introduced into our slab model a layer between SiO2 and the lipid, with the lipid thickness fixed at the value of membrane number two. When we model the confined water layer with the refractive index of bulk water (n = 1.33, k = 0.00), it is not possible to fit the spectroscopic Δ values using any reasonable value of the water thickness (Figure 6D). However, if the refractive index of the confined water is allowed to increase above the bulk value, then a fit to the measured data can be obtained. In this case, the thickness d of the water film is strongly coupled to the refractive index of water such that a thinner water layer yields a higher refractive index but an equally good fit to the Δ values. In the case where n = 1.50, we obtain trivially that d = 1.2 nm, which is the same as the excess thickness of the proximal membrane. Higher values of the refractive index result in a thinner water film. It is well-known that hydration water and water in nanoscale confinements can be structured, resulting in molecular dynamics that differ significantly from that of bulk water.30−32 For example, the dielectric properties for hydration water around proteins is known at low frequencies,33,34 which shows an increase in the real part of the dielectric function compared to that for bulk water. It is possible that a similar effect may explain our indications of a high-n water film next to the substrate, but this should be subjected to further study. Three-component membranes are capable of displaying fluid−fluid phase coexistence of the liquid ordered (lo) and the liquid disordered (ld) phases in a central part of the ternary phase diagram.35,5 We prepared spin-coated supported membranes with two ternary compositions corresponding to frequently used model systems for raft phenomena: T1 = DOPC, DPPC, cholesterol (40%, 40%, 20%) and T2 = DOPC, SM, cholesterol (40%, 40%, 20%). Both membrane composi-

individual membranes in the stacked configuration. We note that, to within experimental uncertainty, the thicknesses of membranes II and III are equal and that their thickness decrease slightly when increasing the temperature from 20 °C to 55 °C. We also note a significantly larger apparent thickness of the first (proximal) membrane, which is adjacent to the solid Si substrate. Previous works by Janshoff et al.28,13,14 have applied IE to extensive thickness measurements of single supported membranes undergoing thermal changes. Their results for changes in membrane thickness above the melting temperature (fluid DPPC) are of similar magnitude to our results for POPC in the fluid phase. However, because our system consists of multiple stacked membranes, we can provide additional information on the effect of the solid support. The fact that the thickness of the second and third membranes is equal is taken as evidence that the influence of the solid support is mainly confined to the proximal membrane. The increase by 1−2 nm in apparent thickness of the proximal membrane, relative to the second and third, could have several explanations. It is unlikely that it corresponds to an increase in actual membrane thickness by this amount because this would be equivalent to a phase transition to the solid/gel state. Although it is known that the phase transition temperature Tm of one leaflet in a proximal supported membrane can be elevated by as much as 10−15 °C,29,28 the solidification of a POPC membrane would require a substantially larger and improbable increase in Tm. A possible effect of substrate roughness which could translate into an effective thickness increase can also be ruled out due to the flatness of our single crystalline substrate. We have examined if a structured water layer between the membrane and the support may explain the apparent thickness increase in the first membrane. Figure 6 shows spectroscopic measurements of double and a single membrane regions (Roi0 and Roi1) in a hydrated POPC film. Analysis of the data by the 1530

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be compared to previous AFM measurements of topographical height differences between lo/ld domains which typically generate values around 0.7−0.9 nm.36 The fact that IE produces approximately twice this value can be explained by a picture of the excess thickness of the lo phase which becomes equally distributed on both surfaces of the membrane, such that AFM detects only half of the absolute thickness difference (insert in Figure 7C). This fact also serves as an indication that thickness differences in supported membranes measured by AFM are not flattened out on the underside due to the support. Along with previous studies of supported membranes using IE, our results demonstrate IE as a highly powerful technique for quantitative and in-depth characterization of model membranes under native and fully hydrated conditions. The technique produces accurate thickness maps while being labelfree and noninvasive. The fact that fluid−fluid domain coexistence is observable in IE opens the possibility for characterizing raft-type phenomena, with or without proteins, in terms of thickness maps. As an example, recent fluorescence microscopy work37 demonstrates that polymer-tethered ternary membranes can be used to answer specific questions concerning the partitioning of transmembrane proteins between the lo and ld phases. Systems of this type could be characterized without labeling molecules, with IE, thereby gaining information on topics such as membrane thickness changes and protein−ligand binding to the membrane surface. In our study, the proximal membrane has effectively functioned as a spacer that decouples the second membrane from the Si wafer. The same effect can be obtained with polymers, and here IE could provide useful information on the actual thickness of the spacer layer.

tions are within the two-phase region at ambient temperature (20 °C) and will exhibit macroscopic domain formation in equilibrium, as previously demonstrated.36 The spin-coated ternary membranes were hydrated and prepared for IE measurements in the same way as for the POPC sample (Figure 5), but after cooling to 20 °C, the samples were stored for 10−15 h prior to measurement to allow resolvable domains to grow by coarsening. Figure 7A and 7B shows ellipsometry thickness maps constructed using the same slab model as for POPC. In both

4. CONCLUSIONS In this work we have measured thickness maps for spin-coated lipid films by imaging ellipsometry (IE). Results show that dry spin-coated films of POPC and DSPC exhibit an ordered multilamellar structure on the support with a proximal monolayer followed by inverted bilayers. The film structure was characterized in terms of thickness histograms which for POPC allow precise counting of the number of bilayers from the support surface. The average film thickness is increasing proportionally to the concentration of the coating solution with nearly the same coating constant for POPC and DSPC. In contrast, the standard deviation of the thickness, which is equivalent to the rms-roughness, shows a considerably higher roughness for DSPC compared to that for POPC. Hydration of the lipid film at elevated temperature produces multiple stacked membranes. On the basis of maps of hydrated membranes in which different numbers of bilayers are exposed, it is possible to differentiate the thickness of the individual bilayers in the stacked membrane system. It is found that the bilayer thickness decreases with increasing temperature. It is also found that the thickness of the proximal membrane appears generally 1−2 nm higher than the thickness of the subsequent membranes. This increase is too large to be assigned to membrane ordering induced by the solid substrate. A more probable explanation for the apparent thickness increase is the presence of a structured water film, with altered dielectric properties, between the membrane and the support. Finally, we have measured thickness maps of liquid-ordered (lo) and liquid-disordered (ld) domains in ternary membranes with compositions DOPC, DPPC, cholesterol and DOPC, SM, cholesterol. The proximal membrane appears homogeneous in

Figure 7. Thickness maps of liquid ordered (lo) and liquid disordered (ld) phase coexistence in the second membrane of two double supported membrane systems. The map in panel A is a system with composition DOPC, DPPC, cholesterol while the map in panel B has composition DOPC, SM, cholesterol (both in the ratio 40%, 40%, 20%). Temperature is 20 °C. Liquid ordered domains are thicker and appear as bright regions. Thickness histograms in panel C constructed from the maps in panels A and B. Histograms are normalized to a range of 1.0 and vertically displaced. Symbols I, IIlo, IIld indicate the location and thickness of the first membrane, where domains are not resolved, and the second membrane in the lo and ld phases, respectively. All scalebars are 20 μm .

cases, sample regions where chosen for IE that contain areas with single and a double membranes. This allows the structure of the proximal and the secondary membrane to be discerned. From the maps and the corresponding histograms in Figure 7C it is clear that lo/ld phase separation is resolved in the secondary membrane while the proximal membrane appears homogeneous. Also, the lo domains in the membrane with composition T2 appear more round in shape than the domains of composition T1. The fact that domains are not resolved in the proximal membrane is attributed to the effect of the solid support which has previously been demonstrated to restrict domain sizes to below the resolution limit of optical microscopy.36 A quantity of interest is the thickness difference between the lo and ld phases which we measure here to 1.69 nm (T1) and 1.89 nm (T2) for the two compositions. This can 1531

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IE, whereas the second membrane displays domains with micrometer lateral size. The thickness difference between the lo and ld domains is approximately twice the height difference measured in AFM topography. This is taken as evidence that the excess thickness of the lo phase is distributed equally on both sides of the membrane.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Danish National Research Foundation is acknowledged for support via a grant to MEMPHYS-Center for Biomembrane Physics. The Carlsberg foundation is acknowledged for financial support. Experiments were carried out on the facilities of DaMBIC - Danish Molecular Biomedical Imaging Center.

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REFERENCES

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