Preparation and Characterization of Solid-Supported Lipid Bilayers Formed by Langmuir Blodgett Deposition: A Tutorial

Transcription

1 This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Cite This: pubs.acs.org/langmuir Preparation and Characterization of Solid-Supported Lipid Bilayers Formed by Langmuir Blodgett Deposition: A Tutorial James Kurniawan, Joaõ Francisco Ventrici de Souza, Amanda T. Dang, Gang-yu Liu, and Tonya L. Kuhl*,, Department of Chemical Engineering, Department of Chemistry, and Department of Materials Science and Engineering, University of California Davis, Davis, California 95616, United States Downloaded via on December 21, 2018 at 22:42:02 (UTC). See for options on how to legitimately share published articles. ABSTRACT: The structure, phase behavior, and properties of cellular membranes are derived from their composition, which includes phospholipids, sphingolipids, sterols, and proteins with various levels of glycosylation. Because of the intricate nature of cellular membranes, a plethora of in vitro studies have been carried out with model membrane systems that capture particular properties such as fluidity, permeability, and protein binding but vastly simplify the membrane composition in order to focus in detail on a specialized property or function. Supported lipid bilayers (SLB) are widely used as archetypes for cellular membranes, and this instructional review primarily focuses on the preparation and characterization of SLB systems formed by Langmuir deposition methods. Typical characterization methods, which take advantage of the planar orientation of SLBs, are illustrated, and references that go into more depth are included. This invited instructional review is written so that nonexperts can quickly gain in-depth knowledge regarding the preparation and characterization of SLBs. In addition, this work goes beyond traditional instructional reviews to provide expert readers with new results that cover a wider range of SLB systems than those previously reported in the literature. The quality of an SLB is frequently not well described, and details such as topological defects can influence the results and conclusions of an individual study. This article quantifies and compares the quality of SLBs fabricated from a variety of gel and fluid compositions, in correlation with preparation techniques and parameters, to generate general rules of thumb to guide the construction of designed SLB systems. INTRODUCTION Cells, their organelles, and other functional volumes are compartmentalized by membranes. 1 Cellular membranes themselves contain hundreds of different constituent molecules, including lipids, sterols, and proteins. These moieties interact laterally to create so-called lipid rafts where the majority of cellular signaling and transport is hypothesized to take place. 2 4 The complexity of cellular membranes, due in large part to the enormous variety of chemical species present and their active function, makes their study challenging. In addition, cellular membranes are constantly in flux and dynamically respond to their local environment. Together, these properties make it daunting to tease out the temporal and spatial variation and even more difficult to correlate structure function relationships. To make headway, various biomimetic or model membrane platforms have been developed. Though vastly simplified, model membranes can mimic essential physical and chemical properties of biological membranes such as membrane elasticity, fluidity, and phase behavior and can provide an appropriate environment for studying protein function. 5 For example, membranes containing mixtures of saturated lipids with high melting points, unsaturated lipids with low melting points, and sterols can form a variety of coexisting phases, and the partitioning of different molecules into these phases has been of particular interest. 6 In an earlier review, Castellana and Cremer 7 described a number of lipid bilayer platforms that have been used as model membrane systems. These platforms allow for the study of a variety of processes. For example, freestanding black lipid membranes are used to study transport across the membrane, while phase behavior and membrane fluidity are frequently studied using giant unilamellar vesicles. 8 Some of the model membrane platforms provide overlapping information, but the specific type of platform dictates the characterization techniques that can be used. Powerful surface-sensitive techniques for measuring membrane interactions, adhesion, and high-resolution scans of membrane topography can easily be used with supported lipid bilayers (SLBs). 9 Freestanding membranes such as unilamellar and multilamellar lipid vesicles enable studies free from the influence of the underlying inorganic support and are useful systems for micropipette mechanical measurements, small-angle scattering, and various imaging microscopies but are incompatible with most high resolution, nanoscopic characterization techniques. SLBs immobilized on clean, smooth, largely hydrophilic surfaces have emerged as a powerful platform for studying Received: October 17, 2018 Revised: November 21, 2018 Published: November 22, 2018 XXXX American Chemical Society A

2 Figure 1. (A) Schematic LB deposition of solid-supported bilayers. (Left) After the substrate has been immersed, lipid is deposited on the air water interface and compressed to the desired surface pressure. (Center) The substrate is drawn out of the subphase perpendicularly to the monolayer at the air liquid interface to deposit the inner leaflet. (Right) To deposit the outer leaflet layer, the substrate is then lowered through the interface. (B) Langmuir Schaefer (LS) method to horizontally deposit the outer leaflet onto a substrate-supported inner monolayer leaflet. (C) Vesicle fusion (VF) on an LB-deposited monolayer. (D) Vesicle fusion directly onto a clean hydrophilic substrate. (E) The top three images are 20 μm 20 μm AFM topographs of 1:1:1 DPPC DOPC cholesterol SLBs deposited via LB or LS onto a 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) inner monolayer LB deposited at a surface pressure of 45 mn/m on mica or by vesicle fusion. The LB and LS techniques on the outer layer were performed at a pressure of 30 mn/m. The bottom images show fluorescence microscopy (FM) of 1,2-dimyristoyl-sn-glycero-3-phosphocholine SLB deposited as an asymmetric (on DPPE) vs symmetric bilayer on a glass substrate using VF. biomimetic membranes, including nano to microscopic phase separation, lipid rafts, and the influence or function of small molecules such as polypeptides and membrane proteins on the nanoscale. The underlying support stabilizes the membrane, and such systems make excellent platforms for sensing applications such as heterogeneous analytical assays for environmental monitoring, drug discovery, and drug testing. 10 However, defects in the SLB could influence the outcomes of individual studies. This invited instructional review focuses on solid-supported bilayer systems primarily formed by Langmuir Blodgett (LB) deposition methods and is organized into three main sections. In the first, methods for preparing SLBs are explained in detail, while the second part of the review covers commonly used characterization techniques of supported membrane systems. The last section of the article characterizes a variety of SLB systems from the nano to the macroscale to elucidate how the lipid composition, phase state, deposition conditions, and substrate influence the resulting SLB. LB deposition conditions to create high-quality, low-defect SLBs are emphasized and validated by characterizing a plethora of SLBs deposited on various substrates to reveal the impact of the surface roughness, lipid phase state, and deposition parameters. PRIMARY METHODS FOR THE PREPARATION OF SOLID-SUPPORTED BILAYERS There are two main methods used to create solid-supported bilayers: Langmuir trough deposition and vesicle fusion. These methods can be used separately or in conjunction to create symmetric or asymmetric membranes with controlled composition. Recently, a third method based on solvent spreading and/or spin coating has emerged as a rapid means to fabricate solid-supported membranes and is briefly described. Descriptions of each deposition technique are provided below. Substrates and the Langmuir Blodgett (LB) Technique. The LB technique, which dates back to 1917, was pioneered by Irving Langmuir 11 for compressing and depositing fatty acid monolayers from the air water interface onto substrates. Katherine Blodgett 12 then expanded the method to multilayers with repeated dipping of a substrate through the air water interface. The LB technique has subsequently been used to measure thermodynamic properties of lipids and other surface-active, water-insoluble monolayer films. The trough, B

3 usually made with a low-surface-energy material such as Teflon, is filled with water or any other subphase such as physiological buffer solution. Widely used substrates include freshly cleaved mica (0001), 13,14 quartz, 15,16 borosilicate glass (microscope slides), 15,17 silicon wafers, and thin films of metal 18,19 or silicon dioxide (SiO 2 ). 20 The quality of the transferred monolayer is greatly enhanced by using ultraclean, hydrophilic substrates with low surface roughness which yield more well-packed, uniform SLBs. Most high-resolution studies use mica or high-quality oxidized silicon wafers because of their low root-meansquare roughness, 0.2 and 2 3 Å, respectively. Recently, a KOH treatment method that greatly reduces the roughness of glass coverslips has been described. 21 To ensure substrate cleanliness, mica should be freshly cleaved right before use. We typically prepare our glass and silicon wafer substrates through methodical coarse to refined cleaning steps: sonication in acetone, sonication in isopropanol, and then rinsing with copious amounts of Milli-Q water to remove contaminants. We then sonicate the substrate in Hellmanex (Hellma Analytics), an alkaline soap for optical components, and again rinse with copious amounts of Milli-Q water. The substrate is then dried using clean nitrogen gas. The clean, dry substrate is subsequently treated for at least 10 min with high-intensity UV ozone and used immediately. UV pen lights can also be used if the exposure time is extended to 30 min. After coarse solvent cleaning, another method for removing organic residue from silica surfaces and increasing the number of silanol groups is with piranha treatment with a mixture of concentrated sulfuric acid and 30% hydrogen peroxide. A common ratio is 3 or 4 parts acid to 1 part peroxide. However, care should be taken as longer than 5 min of exposure can increase the surface roughness. 22 Subsequently, the substrate is rinsed with copious Milli-Q water and dried with clean nitrogen gas. The clean, hydrophilic substrates are immersed in the subphase before spreading lipids on the air water interface in the trough. The lipid spreading solution is prepared in a solvent such as chloroform, hexane, or a mixture of chloroform and methanol. The concentration of the spreading solution is typically between 0.1 and 1 mg/ml. The lipid must fully dissolve in the solvent, and solvent solubility in the trough subphase should be minimal. The choice of solvent and concentration is determined by the solubility of the lipids in the chosen solvent. For example, phosphatidylcholine (PC) lipids fully dissolve in chloroform at room temperature, but phosphatidylethanolamine (PE) lipids require a mixture of 9:1 chloroform methanol for complete dissolution. The spreading solution is dispensed carefully, droplet by droplet, onto the air water interface to create a thin layer of lipids whose hydrocarbon acyl chains anchor the molecules to the interface. For accuracy, an arbitrary solution concentration is typically chosen to achieve a deposited volume of 50 to 100 μl, depending on the surface area of the LB trough and the desired compression of the film. Once the solvent has evaporated, a barrier compresses the lipid monolayer to create a highly compressed two-dimensional film on the air water interface at a predetermined surface pressure. After reaching the target pressure and allowing the compressed film to equilibrate, the solid hydrophilic substrate is vertically drawn out of the water to deposit the inner monolayer. A diagram for bilayer deposition by the LB/LB technique is shown in Figure 1A. Throughout the deposition process, the surface pressure is usually kept constant by compressing the remaining film as the transfer occurs. Once the substrate has cleared the water level, it is resubmerged to deposit the outer monolayer of the SLB. LB/LB deposition can yield a bilayer featuring a single-component lipid or mixtures of lipids with few topological defects. The composition of the bilayer leaflets may be identical or different for the inner and outer monolayer (symmetric vs asymmetric/hybrid bilayer). Some important parameters must be considered in order to achieve a high-quality solid-supported membrane through the LB deposition technique. During deposition, the phase of the lipids greatly affects the resulting monolayer transfer onto the substrate. The lipid phase is dictated by the lipid type, the subphase temperature, and the film s surface pressure (as measured with a Wilhelmy plate). Optimum deposition pressures are usually assessed through the plot of the surface pressure as a function of the area per lipid molecule (Π A isotherm) for the lipid or mixture being deposited. In general, large changes in surface dπ pressure with area,, correspond to better transfers to the substrate. da LB transfer requires strong cohesion of the lipids, which is greater at dπ high pressures and rapid changes in. Low values of, as found in regions of high fluidity or phase coexistence where the pressure changes only modestly with area, correspond to weaker lipid cohesion and result in a variable area per molecule and poor transfer efficacy. At very low surface pressures, little material is transferred from the air water interface to the substrate. 23 = Δ Atrough The transfer ratio, TR, is used to quantify the quality of the Asubstrate monolayer transfer from the air water interface to the substrate. A value of TR = 1 indicates that the surface pressure and therefore area per molecule were maintained from the air water interface to the substrate. 24,25 The dipping speed must also be considered in order to achieve an efficient transfer of lipid from the air water interface to the substrate. A relatively high transfer ratio cannot be obtained if the substrate moves too quickly through the lipid monolayer. In addition, a fast dipping speed sometimes causes a delamination of the inner monolayer rather than deposition of the outer layer. Typically, a dipping speed of between 0.1 and 5 mm/min is chosen in order to obtain a high transfer ratio (TR 1.0). Details of parameters required to obtain an SLB with as few topological defects as possible are discussed in section 4, and Girard-Egrot and Blum provide an in-depth discussion of the LB technique. 26 Langmuir Schaefer (LS) Technique. Vincent Schaefer, in collaboration with Irving Langmuir, first deposited a monolayer of urease on metal and glass plates using a method similar to the LB technique. 27 The LS technique, sometimes called horizontal deposition, deposits a lipid monolayer by stamping the substrate with an orientation parallel to the air water interface through a compressed lipid monolayer (Figure 1B). Typically, the LS technique is used to deposit the outer leaflet of the membrane to create a symmetric or asymmetric SLB with the inner monolayer deposited by LB. The LS technique should be used to create the SLB in cases where the physiochemical interaction of the inner layer with the substrate is insufficient to ensure stability. In other words, LS is particularly useful in situations where the inner monolayer can delaminate from the substrate during vertical LB deposition. 28 The lipid phase state is also important for the LS technique. Gerelli et al. determined that fluid leaflets are mixed during the LS deposition presumably due to mechanical shock. 29 To maintain leaflet asymmetry, which is frequently used to study lipid flip-flop rates, lipids should be deposited in the gel phase. Dipping speed is not as crucial for achieving a quality bilayer using the LS method. However, the orientation (that is, how level the substrate surface is relative to the water surface) is critical for successful LS deposition. When the substrate is at an angle, film material can be pushed away, leading to a less-well-packed transfer. A detailed comparison of SLBs fabricated with the LB/LB technique versus the LB/LS technique is provided in section 4. Vesicle Fusion (VF) Technique. Many SLB studies use the vesicle fusion method to prepare bilayers on substrates because of its ease and simplicity. To create an SLB, small unilamellar vesicles are incubated on a clean hydrophilic surface to create a symmetric bilayer (Figure 1D). Vesicles can also be incubated with an LB-deposited monolayer of a different composition to make asymmetric membranes (Figure 1C). 33 Vesicle fusion relies on the instability of vesicles interacting with the support and attractive interactions of the vesicles with the support (including already-fused membrane regions) to yield spontaneous SLB formation. 17,34 Osmotic stress, the addition of salts and divalent ions, and temperature cycling can also be used to aid SLB formation and SLB coverage of the substrate and break any absorbed, intact vesicles However, the quality of SLBs deposited by VF or LB/VF is often more variable compared to that of LB/LB or LB/LS deposited bilayers (Figure 1E) VF is less reproducible and may exhibit incomplete surface coverage with holes or defects as well as adsorbed, nonfused vesicles In LB or LS techniques, the monolayer surface pressure is precisely controlled, which generally leads to fewer topological defects d d Π A da C

4 Figure 2. Fluorescence microscopy images of 2:2:1 DPPC DOPC cholesterol SLB formed on a microscope slide using vesicle fusion of 100 nm SUV solution in 0.5 mm NaNO 3. (A) SLB after 45 min of incubation and 3 min of rinsing with Milli-Q water at 25 C. Numerous, large membrane topological defects can be seen. (B) Membrane topological defects shrank with 15 min of heating at 45 C. The bright spots show where vesicle/ tubules have started to form. (C) Longer heat application resulted in the formation of long tubules and vesicles, resulting in a significant loss of lipids from the SLB into solution. (D) A representative 25 μm 25 μm AFM topographic image of a 2:2:1 DPPC DOPC cholesterol SLB formed on mica using vesicle fusion appears to be homogeneous. (E) At higher magnification, numerous vesicles are observed to populate the membrane surface. (F) The same area as in D with a central 1 μm 1 μm area of film shaved to enable height measurement and assess packing through thickness measurement. or variations in the SLB by comparison. Regarding high-resolution membrane structure, parity across techniques is possible. Watkins et al. 24 established that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes deposited by vesicle fusion mimic deposition by the LB/LS technique at surface pressures of 38 ± 3 mn/m. The first step in performing vesicle fusion is the preparation of the vesicle solution. A small amount of concentrated lipid in an organic solvent is prepared in a glass vial or similar container. The solvent is then evaporated by a stream of nitrogen while rotating the vial to leave a thin lipid layer on the walls of the container. The amount of lipid solution needed depends on the required volume of the vesicle solution. In general, the concentration of the vesicle suspension used for SLB formation is between 0.2 and 1 mg/ml. After drying with nitrogen, the vial is placed in a vacuum for at least 2 h to fully remove all solvent before hydrating the lipids to create the vesicle suspension. Applying a vacuum is essential, as any solvent present upon addition of water will disturb vesicle formation. The dried lipids are then hydrated with water or buffer solution, and the suspension is vortex mixed to obtain multilamellar vesicles of heterogeneous sizes. Freeze thaw cycles can be used to break the multilamellar structures into unilamellar vesicles. Small unilamellar vesicles (SUVs, diameter 100 nm) are preferable for vesicle fusion because they more readily absorb/rupture on the substrate to form an SLB. To make small, uniform SUVs, the vortex mixed vesicles are probe-tip sonicated (typically for 1 to 2 min) or extruded through a polycarbonate membrane multiple times with the solution temperature above the lipid or lipid mixture melting point. Extrusion is the preferred method, as probe tip sonication can release contaminant titanium particles into the vesicle solution, though these can be removed by centrifugation or an extra filtration step. The substrate is then incubated with the freshly prepared vesicle suspension for at least 20 min based on surface plasmon resonance and quartz crystal microbalance studies of the time required to reach saturation. 43 Excess vesicles can be washed away after the incubation. Vesicle fusion usually results in a bilayer with many topological defects, adhered vesicles, and suboptimal surface coverage However, there are a number of ways to increase the quality of vesicle fusion SLBs. 17,40,44 Typical preparation methods include incubating a room-temperature substrate with a warm SUV solution (in general, the vesicle solution is always above the melting point of the lipids), freeze thaw cycling, and rinsing with water at an elevated temperature or applying an osmotic shock by rinsing with a solution of a different salt concentration to obtain higher bilayer surface coverage. In particular, the incubation must be performed with a fresh SUV solution because small unilamellar vesicles are unstable and will readily fuse to the clean hydrophilic substrate to make a symmetric SLB or will fuse to a monolayer to create an asymmetric SLB. 45,46 Freeze thaw cycling will rupture adsorbed vesicles through the formation of ice crystals, so they can deform and cover the substrate. 47 Recently, fusogenic peptides have been incorporated to improve vesicle fusion. 48,49 After incubation, excess and physisorbed vesicles are removed by exchanging the incubation solution with a vesicle-free buffer solution or water. If the rinsing solution has a different salt concentration than the vesicle solution, then the concentration gradient creates osmotic flow that swells or shrinks and ruptures any excess vesicles stuck to the membrane or substrate and thus results in a more uniform supported membrane. This washing step is usually repeated a few times to ensure the removal of excess vesicles. Care must be taken to ensure that the SLB is not exposed to air, which will disrupt the bilayer. After the SLB is deposited on the substrate, thermal cycling/annealing can be done to increase the surface coverage after incubation. 38 However, the heating and cooling cycle must be done cautiously to prevent excessive loss of lipids into the subphase (Figure 2A C). For comparison, the bottom panel (Figure 2D F) shows higher-resolution AFM topographs of SLBs of the same composition, 2:2:1 DPPC DOPC cholesterol, formed by vesicle fusion on mica. Spin Coating, Spreading Techniques, and Rapid Solvent Exchange. Spin coating and spreading are quick and easy techniques for the deposition of solid-supported lipid membranes. In spin coating, a lipid solution with concentration ranging between 0.25 and 5 mm in a volatile solvent that wets the substrate is deposited on a clean substrate. After spreading, the substrate is rapidly accelerated to a certain rotational speed (e.g., 2000 rpm for 2 min) to quickly remove the solvent, leaving a thin, dry lipid film. In the spreading technique, one microliter of lipid solution with a similar concentration range is deposited on a clean substrate and the solvent is allowed to evaporate. 50,51 Once a thin, dry lipid layer is formed on top of the substrate, the sample can then be partially or fully hydrated to create a stack of lipid membranes. Mennicke and Salditt 52 described parameters for the spin-coating process in detail, such as lipid solution concentration and rotational speed, in order to deposit multiple D

5 bilayers (up to 22 layers). Excess floating bilayers can subsequently be removed by a fluid jet to leave a single supported bilayer in the treated region. 53 The fluid jet is typically water or buffer sprayed onto the substrate from a syringe or Milli-Q water dispenser. While this technique can be used for solid-supported membrane preparation, it is more suited for creating multiple bilayers on a substrate. This is due to the numerous topological defects and low stability of multilayers in bulk water as characterized by X-ray scattering and other methods Rapid solvent exchange or solvent-assisted lipid bilayer (SALB) formation utilizes the phase behavior of lipid/water/isopropanol mixtures to drive the formation of the SLB. 56 Although other miscible alcohols can be used (e.g., methanol, ethanol, and n-propanol), the most uniform SLBs were formed using isopropanol. 57 A range of lipid and isopropanol concentrations have been demonstrated to work, ranging from 0.5 mg/ml lipid in pure isopropanol followed by water/ buffer exchange to 2 mg/ml lipid in a 50:50 volume solution of water isopropanol followed by water/buffer exchange. The main requirement is during the incubation period, in which the lipids in the isopropanol/ water mixture should be below the micelle-to-vesicle transition point. Upon increasing the relative water content, the critical micelle concentration of the lipid is lowered and the lipid substrate interaction drives the formation of the SLB. Similar lateral diffusivities were obtained for vesicle fusion and SALB-formed SLBs; however, care must be taken during water washing steps to ensure the elimination of residual alcohol. A distinct advantage of the solvent exchange method is that no specialized equipment is required. In addition, the formation of SLBs with high cholesterol content can be obtained, which is especially challenging with VF methods. 58 CHARACTERIZATION OF SOLID-SUPPORTED BILAYERS A solid-supported membrane created by any of the methods described above can be characterized by a variety of techniques. Characterization techniques provide information on SLB properties, and the results from one technique can corroborate data collected by another method. Commonly used characterization techniques are described below. Fluorescence Microscopy (FM). Fluorescence microscopy is a basic but powerful technique that relies on doping with a small amount of fluorescent lipids (usually 0.25 to 1 mol %) to characterize solid-supported membrane systems. White light is directed through a band-pass (excitation) filter that allows only light of a specific wavelength range to pass through and is directed to the sample using a dichroic mirror. To reduce noise and fluorescent contributions from off-target molecules in the sample, the wavelength range is selected to match the absorption wavelength of the fluorescent dopant. Illumination excites the dye, resulting in the release of light with a longer wavelength that passes back through the dichroic mirror and an emission filter into the detector. FM enables lateral organization, including phase separation and domains of the SLB to be studied on the macroscale, since a given fluorescent dye will often partition preferentially into one phase more than into the other. FM also permits the analysis of membrane fluidity through fluorescence recovery after photobleaching (FRAP). FRAP enables the determination of the diffusion coefficient of the fluorescently labeled lipid and the mobile and immobile fractions of the SLB. High-quality SLBs have uniform diffusivities and low immobile fractions. Loren et al. and Rayan et al. have published recent reviews on FRAP. 59,60 In a typical measurement, a small region of the membrane is photobleached by exposing the region to a high-intensity light source. The rate of fluorescence recovery in the bleached spot is then used to calculate the lipid diffusion coefficient while the recovered intensity compared to the initial intensity is used to determine the fraction of the SLB that is mobile. Figure 3 shows an example FRAP measurement and fluorescence recovery time curve from an SLB system. The Figure 3. Schematic of the FRAP technique showing a typical fluorescence intensity curve of a bleach spot as a function of time. The diffusion coefficient is determined by fitting a model to the intensity recovery curve. 59,60 The difference in the recovered intensity with time, I t 0, vs the initial intensity, I 0, provides a measure of the fraction of the SLB that is mobile or able to diffuse. diffusivity is greatly impacted by the phase state of the membrane. Values typically range from on the order of 5 μm 2 /s for fluid-phase-supported membranes to 0.1 μm 2 /s for liquid-ordered (L 0 ) phase-supported membranes to 10 3 μm 2 /s for gel-phase-supported membranes. 15,61 There are additional configurations of fluorescence microscopy that are used to characterize SLBs. For example, Fo rster resonance energy transfer (FRET) can be used to observe microdomains in a lipid raft 62 and the interaction between proteins in the membrane. 63,64 Total internal reflection fluorescence (TIRF) microscopy and two-photon excitation fluorescence microscopy (P2FM) can be utilized to study the directional alignment of the lipids in a gel-phase domain. 65,66 Confocal fluorescence correlation microscopy, an improvement upon traditional FM, increases the resolution of lateral diffusivity measurements and can produce a three-dimensional structural scan of the solidsupported membrane if there are features protruding out of the membrane plane. 67 The fluorescent dyes used in FM have several key characteristics, such as headgroup or acyl chain labeling, partitioning preference into more ordered or disordered phases, their excitation/emission spectrum, quantum yield, and lifetime, which must be carefully considered against the demands of the experiment. For example, FRET requires two fluorescent dyes such that the emission spectrum of one dye overlaps the excitation spectrum of the other dye. FRAP necessitates a dye with a robust quantum yield and lifetime that is still easy to photobleach. Berezin and Achilefu qualified various fluorescence dyes in detail, including their spectra and lifetime. 68 Online tools from fluorescent dye vendors also aid in determining the best dye for each specific use. 69 One caution is that the presence of fluorescent probes can shift the miscibility and phase transitions in some systems Atomic Force Microscopy. Atomic force microscopy (AFM) scans a sharp tip over samples and provides a powerful means to characterize solid-supported bilayer systems due to its E

6 Figure 4. (A) AFM topograph image (45 μm 45 μm) of an asymmetric SLB composed of an outer 3:7 DPPE DOPC on an inner DPPE monolayer on mica(0001) revealing a gel-phase DPPE domain (bright) island in a fluid phase (gray) of primarily DOPC in the outer leaflet, punctuated by defects (dark spots). (B) The cursor profile as indicated in A shows the height difference between gel- and fluid-phase regions. high-resolution, label-free nature and versatility to work in various environments, including ambient, liquid, and culture media. 73,74 The local membrane structure and properties have been clearly revealed by AFM, including topography, domain size, layer thickness, friction, adhesion, and viscoelastic properties. 38,41,75 78 SLBs prepared with various previously described deposition techniques can be imaged using AFM under a defined medium. Acquiring AFM images of monolayers in an ambient or nitrogen environment is relatively straightforward. For imaging in water or liquid media, cautions are necessary when designing and constructing sample holders to minimize sample perturbation and maintain the SLB s structural integrity. 79 Two raster scan imaging modes are primarily used to characterize the structure and morphology of SLBs: tapping and contact mode. 38,80 Contact mode with soft cantilevers typically yields high-resolution images for gel phases, while tapping mode is necessary for liquid phases or layers with hydrophilic termini, which tend to have high adhesion to Si and Si 3 N 4 AFM tips. In contact mode, the tip apex is in firm contact with the surface throughout the scan, while maintaining a set load. 81 In this case, topographic and lateral force images are acquired simultaneously for both trace and retrace scan directions. Note, the lowest imaging force should be used to avoid the compression of the SLB. The topographic images reveal the surface contour, while the lateral force images show local functionality, such as hydrophilic versus hydrophobic domains. In tapping mode, the cantilever tip is modulated at the resonance frequency of the cantilever, whose amplitude would decay as the tip approaches the surface. The control electronics maintains the tip surface separation based on set damping, e.g., 80% of the initial amplitude. Typically, topographic and phase images are acquired simultaneously. The former reveals feature heights, while the latter is related to the tip surface interaction, which again depends on the local functionality and mechanics. In addition to imaging, force versus distance measurements can be acquired to reveal tip surface interactions at chosen locations. This spectroscopic measurement is also known as a force deformation profile or curve, from which the repulsive and adhesive forces between the tip and film can be extracted. 82 With designed functionalization of the AFM tip, the adhesive force may be correlated to the hydrophilicity of the surface feature, DNA hybridization force, or specific interactions such as ligand receptor binding For AFM imaging of SLB systems presented in this work, tapping mode was frequently used at a speed of 20 μm/s, at 80% of the initial amplitude (45 nm). Contact mode imaging was done at the same speed, but under a load of 6.2 nn. In both cases, the tip was an AC240 (Olympus, Japan) with a spring constant of 1.7 N/m. Figure 4 shows an example AFM topograph of an SLB acquired using tapping mode. The SLB consisted of an inner monolayer of 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE) and an outer layer of 3:7 DPPE 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids. At room temperature, DPPE (T m =63 C) and DOPC (T m = 20 C) phase separate. The AFM topography image clearly shows phase separation in the SLB system. The gel-phase DPPE domain in Figure 4 is about 1.5 nm taller than the surrounding DOPC fluid phase. Surface Forces Apparatus (SFA). Various direct forcemeasuring techniques have been developed which enable the measurement of the interaction between membranes with various geometries. 83,86 Using a variety of (interchangeable) force-measuring springs, the SFA can directly measure the full force profile between two SLBs in liquid at the angstrom (0.1 nm) level with a force resolution of 10 8 N. The solution in the SFA can be exchanged in situ to study SLB interactions under different conditions. 14,20,87,88 SFA characterization is very powerful because it is possible to unambiguously determine the interaction force profile, thickness, and refractive index of the SLBs as well as observe structural rearrangements when the SLBs are in contact. 13,88 Figure 5 shows example interaction force distance profiles between liquid-ordered SLBs, which reveal nanoscopic structural rearrangement of the SLB in contact. A positive force indicates repulsion and a negative value indicates adhesion between the SLBs. The measured profile is the sum of any interactions present such as electrostatic, steric, van der Waals, depletion, hydration, and hydrophobic forces. By careful experimental design, the contributions of the various interactions can be separated and quantified. The substrate used in SFA is usually back-silvered, molecularly smooth mica that is glued onto a cylindrical disk (radius of 1.5 cm). Once the mica is glued onto the disk, the desired SLB can be deposited, typically using the more controlled LB technique. After membrane deposition, the surfaces are transferred under water into the SFA and the solution is saturated with lipid of the same composition as the membrane to minimize the desorption of lipids from the surfaces. The two surfaces are positioned in a cross-cylindrical configuration that is locally equivalent to a sphere near a flat surface or two spheres close together. White light is passed through the opposing surfaces, and the emerging beam is focused onto the slit of a grating spectrometer. The silver layer on each disk partially transmits light directed normally through the surfaces, which constructively interferes and produces fringes of equal chromatic order (FECO). The distance between F

7 Figure 5. Force distance profiles between asymmetric SLBs composed of an outer 1:1:1 DOPC DPPC cholesterol monolayer on an inner DPPE monolayer in 0.5 mm NaNO 3 solution. D = 0 is defined as the contact between bare mica surfaces. The inset shows four successively measured force profiles while allowing a longer contact time between each distance displacement. The inward shift to smaller separations (decreasing membrane thickness) and greater adhesion is due to lipid rearrangements in the contacting region {(, ) approach; (leftpointing, right-pointing ) separation}. Reproduced with permission from ref 87. Copyright 2014 American Chemical Society. the two surfaces can be adjusted using a motor connected to the lower surface or a piezo connected to the upper surface. The separation between the surfaces is measured by monitoring the wavelength of the FECO. The lower surface is supported on a double cantilever spring with a known spring constant {typically around (1 2) 10 5 mn/m for SLBs}. Both repulsive and attractive forces can be measured, and the force profile can be obtained over a large distance regime under quasi-static conditions. Once the force F as a function of distance D is measured for the two surfaces (of radius R), the adhesion or interfacial energy E per unit area can be calculated using the Derjaguin approximation: E = F/2πR. Thus, for R 1 cm and given the measuring sensitivity in F of about 10 8 N, the sensitivity in measuring adhesion and interfacial energies is approximately 10 2 mj/m 2 (erg/cm 2 ). In addition to measuring interaction forces between SLBs, 14,20,87 90 the SFA has been used to probe the interaction between SLBs and substrates, 91 the hemifusion of SLBs, 89,92 membrane-mediated receptor ligand interactions, 93,94 the refractive index and thickness of supported monolayers and membranes, 13,14 and the interactions of adsorbed vesicle layers. 95,96 X-ray and Neutron Reflectivity. X-ray and neutron reflectivity are powerful surface-sensitive characterization techniques which provide information such as the thickness, density profile, and roughness down to the atomic scale for a substrate-supported thin film. Reflectivity, R, isdefined as the intensity ratio of X-rays or neutrons (hereafter referred to as particles) elastically and specularly scattered from the surface relative to the incident particle beam. The reflectivity is measured as a function of the wave-vector transfer, 4 q = π sin θ z, perpendicular to the interface, where θ is the λ angle of the beam with respect to the sample and λ is the wavelength of the particle beam. When measured this way, the reflectivity curve contains information regarding the average scattering length density of the sample normal to the interface and can be used to determine the concentration of atomic species at a particular depth in the film. Detailed descriptions of reflectivity measurements of SLBs can be found in the literature An example X-ray reflectivity profile from a DPPC SLB LB/LS deposited on quartz is shown in Figure 6A. 16 The visible fringes in the reflectivity profile arise from interference between particles reflected from the membrane solution interface and membrane substrate interface. The amplitude of the fringes is due to the scattering length density (SLD) contrast among the substrate, lipid headgroups, and acyl 2 chains of the bilayer, while the fringe spacing, Δq π z d,is layer related to the thickness of the film (d layer ). From the measured reflectivity profile, the SLD, thickness, and roughness of the Figure 6. (A) Reflectivity data obtained from neutron and X-ray scattering measurements of solid-supported DPPC bilayers on quartz. The X-ray data covers a much larger q range, yielding higher resolution. The solid line is the fit to the data corresponding to the scattering length density profile shown in B as the darkest shaded curve. (B) Density profile of DPPC bilayers deposited using LB/LS (LBS) and the VF method obtained. From the electron density distribution, the thickness of the DPPC SLB and the number of water molecules associated with the bilayer and substrate were inferred. The dark shaded curve corresponds to the data shown in A. The different bands represent the variation in the structure of the SLB by displaying the scattering length density profile from a minimum of four independent samples including at least four measurements from different regions of each sample. Adapted with permission from ref 16. Copyright 2009 American Physical Society. G

8 various layers can be determined by modeling the expected SLD profile and iterating to minimize the difference between the measured reflectivity profile and that obtained from the modeled SLD profile. However, as the majority of reflectivity measurements provide only intensity information, the structural information of interest is indirectly contained within the reflectivity data. The transformation of the data from inverse space to real space, in the absence of phase information, is mildly ill-posed, and multiple solutions can be obtained. 107,108 Limiting the possible solutions through constraints based on the known chemical identities of the layers, expected thickness, and other information is extremely helpful. The corresponding scattering length density profile of the DPPC bilayer fitted to the measured reflectivity profile is shown in Figure 6B. There are a number of key differences between neutron and X-ray scattering. Neutrons can penetrate large sample volumes and do not damage the sample as the beam is scattered by atomic nuclei. Selective contrast can be achieved by the deuteration of lipids/proteins and solvent contrast variation. 33 On the other hand, X-ray sources are much more brilliant. Because the beam is scattered by electrons in the sample, significant energy is deposited in the sample, which can quickly degrade organic samples. 109 Although contrast is limited to the electron density of the film, the high intensity of X-rays allows much more rapid and higher-resolution measurements, as shown in Figure 6A. The intensity also enables diffraction measurements from ordered monolayers and SLBs, yielding molecular details such as unit cell parameters, tilt angle, coupling between leaflets, and lateral coherence lengths from the off-specular diffraction signals (grazing incidence X-ray diffraction or GIXD). 16,24,104 SLBs for neutron and X-ray reflectivity are prepared on smooth, ultrapolished single-crystal substrates (e.g., quartz, oxidized silicon, sapphire) using LB LS or vesicle fusion. The sample is mounted in a holder, and the neutron/x-ray beam is reflected off of the substrate through the SLB, which sits in a thin water layer. The scanning area for neutron reflectivity is approximately cm 2 ; a significantly smaller area of about 0.1 cm 2 can be used for X-ray reflectivity. The scattering signal and the quality of the data is dependent on multiple factors, such as interfacial roughness (quality of the substrate and SLB), the contrast of the various layers, and the level of incoherent scattering from the bulk liquid. For the purposes of obtaining basic structural information about an SLB with high resolution, higher fluxes of the X-ray source coupled with faster measurement times make X-ray reflectivity generally superior to neutron reflectivity. On the other hand, neutron reflectivity can take advantage of deuteration to enable specialized measurements such as the flip-flop of lipids between the inner and outer leaflets of a bilayer. 29 Details about neutron scattering characterization methods, including a discussion of advantages, limiting factors, recent work on SLB characterization, and an extension of the use of neutron scattering on various platforms can be found in several recent publications. 98,110,111 Quartz Crystal Microbalance (QCM). QCM can be used to track the quantity of absorbed mass on a solid with time. The quartz crystal oscillates at a frequency that is dependent on its mass. When films are deposited/adsorbed on the crystal, the frequency of the oscillation decreases and the change in the oscillation frequency can be correlated to a change in mass. The thickness of the deposited/absorbed layer can be calculated using an equation developed by Sauerbrey. 112 In the case of SLBs, QCM measurements have been used to study the process of vesicle fusion as a function of time and are becoming a standard technique for optimizing the conditions for SLB formation. 34,113 In-depth studies of the mechanics of vesicle fusion with various lipid mixtures have been performed to determine parameters that affect vesicle adsorption kinetics on various substrates QCM can also be coupled with other techniques such as AFM and surface plasmon resonance (SPR) to visualize the various stages of SLB formation via vesicle fusion. 117,118 In addition to obtaining information on the kinetics of vesicle fusion, QCM can be used to probe the interaction between various lipids with different types of substrates 119 and the interaction between deposited SLBs and proteins 120 or nanoparticles. 121 An in-depth discussion of the QCM technique, including its diverse uses extending beyond SLB systems, can be found in works by Cooper and coworkers. 112,122 IMPACT OF DEPOSITION CONDITIONS ON THE QUALITY OF SUPPORTED LIPID BILAYERS In most cases, a clean, stable, well-packed membrane provides a good platform for studies involving SLBs. Membrane topological defects, which span the outer monolayer or in some cases penetrate the inner monolayer, are typically present when SLBs are deposited via any commonly used preparation technique (hereafter referred to as membrane defects). In most cases, these defects are nanoscopic and not visible through optical microscopy, such as fluorescence or confocal microscopy. 23,92,123,124 These nanoscopic features can be resolved by high-resolution AFM imaging and can play an important role in altering the structure, properties, and interactions of the SLB. In this final section, qualitative and quantitative comparisons of SLBs deposited under different conditions are provided and discussed to optimize procedures and conditions toward constructing continuous SLBs with closely packed molecules and few defects. Because vesicle fusion SLBs are more variable, LB deposition methods are emphasized. Vesicle fusion can yield high-quality SLBs, but the interplay between the vesicle substrate interaction, vesicle size, composition, substrate properties, and especially solution conditions such as the ionic strength and temperature all must be carefully considered and optimized to yield high-quality SLBs. Moreover, complementary characterization techniques must be used to fully characterize the SLB. Namely, small defects and adsorbed/unfused vesicles can be difficult to detect. A number of papers have addressed these aspects. 113,125,126 Further, many of the important experimental deposition parameters described for LB deposition in the next section also impact vesicle fusion SLB quality such as the substrate roughness and lipid phase state, while other conditions such as the surface pressure during deposition, packing properties of the inner monolayer leaflet, and other preparation conditions are controllable and explored via LB deposited films. These parameters are probed quantitatively by transfer ratio measurements conducted with the LB deposition technique and high-resolution AFM topographs to reveal the heterogeneity and coverage of the resulting SLB. The characterization of the SLBs moves from the macro/micro to the nanoscale in the following subsections. Quantification of SLB Quality by Transfer Ratio Experiments and the Selection of Deposition Pressure. One of the most important Langmuir deposition parameters that dictates the quality of the resulting SLB is the surface pressure during deposition. The surface pressure area (Π A) isotherm depends on the lipid mixture and temperature and can be used to select the desired lipid packing (area per molecule) of H

9 Table 1. Summary of the Transfer Ratio for Various Lipid Mixtures Deposited with the LB Technique a lipid Π (mn/m) deposited on phase transfer ratio inner layer DTPC 30 glass fluid ± 0.7 DMPC 30 glass transition (gel to fluid) ± 0.8 DPPC 45 glass gel 89.9 ± 1.0 DPPE 45 glass gel 92.0 ± :8 DPPC chol 30 glass transition (gel to liquid-ordered) ± 0.3 1:1:2 BSM POPC chol 30 glass liquid-ordered ± 3.2 DTPC 30 mica fluid 93.4 ± 1.3 DMPC 30 mica transition (gel to fluid) 95.7 ± 1.1 DPPC 45 mica gel 97.2 ± 1.8 DMPE mica gel ± 0.3 DPPE 45 mica gel 98.2 ± :8 DPPC chol 30 mica transition (gel to liquid-ordered) 88.8 ± 3.0 1:1:2 BSM POPC chol 30 mica liquid-ordered 80.1 ± 1.8 DTPC 30 silicon wafers fluid 93.7 ± 2.0 DMPC 30 silicon wafers transition (gel to fluid) 96.5 ± 0.5 DPPC 45 silicon wafers gel 96.2 ± 0.7 DPPE 45 silicon wafers gel 95.9 ± 1.7 outer layer (substrate: mica) DTPC 30 DTPC fluid 61.7 ± 5.6 DPPE 45 DTPC gel 96.2 ± 0.4 DMPC 30 DMPC transition (gel to fluid) 75.1 ± 1.8 DPPC 45 DPPC gel ± 0.6 DTPC 30 DPPC fluid 98.4 ± 0.7 DTPC 30 DPPE fluid 97.5 ± 1.6 DMPC 30 DPPE transition (gel to fluid) 96.0 ± 1.6 DPPC 45 DPPE gel 90.5 ± :8 DPPC chol 30 DPPE transition (gel to liquid-ordered) 95.8 ± 0.7 1:1:1 DPPC POPC chol 30 DPPE liquid-ordered 96.0 ± 1.4 1:1:2 BSM POPC chol 30 DPPE liquid-ordered 98.6 ± 1.1 DTPC 30 dried DPPE fluid 99.0 ± 2.1 DMPC 30 dried DPPE transition (gel to fluid) 97.3 ± 0.5 DPPC 45 dried DPPE gel 97.7 ± :8 DPPC chol 30 dried DPPE transition (gel to liquid-ordered) 97.8 ± 0.8 1:1:1 DPPC POPC chol 30 dried DPPE liquid-ordered 98.3 ± 0.3 1:1:2 BSM POPC chol 30 dried DPPE liquid-ordered 99.5 ± 1.8 a In all cases, TR = Δ Atrough Asubstrate with no rescaling for TR < 1 with the inner leaflet. the deposited monolayer. Typically, deposition using the LB or LS technique is conducted in which the slope of the Π A isotherm curve is at its steepest ( d Π, largest change in the surface d pressure with small shift in area A per molecule) and the monolayer is still stable (showing little loss in area with time prior to deposition). 26 The surface pressure is held constant during the deposition by decreasing the area of the monolayer film as material is transferred from the air water interface to the substrate. Because the change in pressure with area is small, monolayers cannot be transferred well at low surface pressure or in coexisting regions. For example, Bassereau and Pincet investigated the impact of deposition pressure in SLBs composed of inner leaflets of DMPE and outer leaflets of DOPC formed by LB/LB deposition on mica. When the outer DOPC monolayer was transferred at low surface pressures (π 25 mn/m), holes were found in the SLBs. The size and surface coverage of the holes increased as the DOPC surface pressure decreased. Bassereau and Pincet were able to correlate the formation of holes with the desorption of the inner DMPE monolayer from the substrate back to the air water interface during transfer of the outer, poorly packed DOPC monolayer. The desorption of DMPE was further enhanced at slow dipping speeds. Similar results were found with symmetric SLBs composed of pure DOPC and DMPE, demonstrating the need for high surface pressure and high lateral cohesion of the lipids during LB deposition. Subsequently, Benz and co-workers investigated the morphology and interactions of SLBs as a function of deposition conditions of the outer monolayer. 92 Their work clearly demonstrated that defects were present in the outer leaflet and that the density and size of the defects increased with decreasing deposition pressure. At ultralow outer monolayer deposition pressures (4 mn/m), membranespanning holes were observed due to the rearrangement of the inner leaflet. As described in section 4.2, membrane-spanning holes in SLBs are minimized by using well-packed inner and outer leaflets deposited at high surface pressures, consistent with the requirement of strong lateral cohesion between lipids in the deposited monolayers for the formation of high-quality SLBs. Gel-phase inner leaflets such as DPPE and DPPC also minimize the formation of membrane holes. Once the pressure for the deposition is selected, the quality of the deposited inner and outer monolayers can be quantified on the macroscale based on a transfer ratio measurement. The transfer ratio is defined as the ratio of the area of lipids removed from the air water interface to the substrate area coated during the deposition (as described in section Substrates and the LB technique). In all cases here, transfers were done with the surface I

10 pressure held constant by computer control during the deposition. Deposition speeds were typically 1 mm/min. A systematic study of deposition speed with respect to SLB transfer quality remains to be done. Table 1 summarizes the transfer ratios of various lipid mixtures deposited on different substrates with the LB technique. The impact of specific conditions on TR is highlighted below. Substrate Roughness. To quantify the effect of substrate roughness on the deposition quality, a comparison of mica, silicon wafers, and microscope slides (borosilicate glass) was carried out. In general, the transfer of lipid monolayers onto glass slides or coverships resulted in higher transfer ratios than onto mica or silicon wafers. This is because of differences in substrate roughness. As measured by contact mode AFM, glass slides have a significantly larger surface roughness (root-mean-square roughness, RMS = 8 10 Å) than do silicon wafers (3 4 Å) or mica (0.2 Å), creating a larger effective surface area on the glass. The transfer ratio of pure, fluid-, and transition-phase lipids increased from about 95% surface coverage on mica to 100% or greater on glass. Similar observations were seen for mixed lipid systems containing cholesterol, where the transfer ratio increased from less than 90% to about 100%. The only cases where the transfer onto mica was greater were for gel-phase DPPE and DPPC. This is likely due to the greater stiffness of the gel phase and lateral cohesion of the lipid monolayer, which prevents good conformity and physisorption onto rougher glass slides. Conversely, mica and silicon s ultrasmooth surfaces are especially well suited for the transfer of gel-phase monolayers. When the monolayer contains cholesterol or exists in fluid or transition phases, it can conform to the roughness of the glass substrate and result in a higher transfer ratio. However, as shown through high-resolution AFM topography scans in section 4.2, the SLB is of lower quality on rougher substrates. We also examined the transfer ratio of various lipid mixtures onto DPPE and DPPC monolayers on mica, glass slides, oxidized silicon, and quartz. However, the inner monolayer always delaminated when attempting to LB deposit the outer leaflet onto SiO 2 surfaces, reinforcing the need for the LS method with these substrates. 28 Unfortunately, measuring TR with LS is very challenging due to the disruption of the remaining film during transfer. As a result, outer-layer transfer ratios are only reported with mica substrates. Lipid Phase State: Impact of Deposition Pressure. The lipid monolayer phase state and substrate roughness influenced the transfer ratio of the inner leaflet as described above. Here, we compare how the phase state of the inner leaflet affected the transfer ratio of the outer leaflet. At room temperature, the transfer ratios of fluid onto fluid (DTPC on DTPC) and transition phase onto transition phase (DMPC on DMPC) were below 80%. However, the transfer of gel onto gel (DPPC on DPPC), gel onto fluid (DPPC on DTPC), and fluid onto gel (DTPC on DPPC) were significantly higher. Interleaflet mixing and desorption of some of the deposited lipid in the inner monolayer disrupts the outer-layer transfer in more fluid- or transition-phase depositions. 23,29 When at least one of the layers is in the gel phase, interleaflet mixing is diminished and higher transfer ratios are obtained. In general, gel-phase lipids provided a good base for the LB deposition of outer monolayers of various phases. As detailed in section 4.2, gel-phase base monolayers have very few defects, resulting in more complete SLB formation. We also found that the outer monolayer transfer was slightly enhanced when the inner leaflet was dried by comparing transfer ratios onto freshly deposited DPPE inner monolayers and monolayers that were dried overnight (relative humidity <25%). If the outer-layer deposition was delayed for at least 8 h after the deposition of the inner monolayer, then the transfer ratio of the outer leaflet increased for a number of different lipids. Drying the inner monolayer helps to remove trapped moisture between the monolayer and substrate, leading to reduced desorption during the transfer of the outer monolayer. Finally, the higher transfer ratio of symmetric, gelphase DPPC SLBs is due to interleaflet lipid coupling. In a recent X-ray scattering study, it has been shown that symmetric DPPC bilayers couple across the membrane and are more condensed than in their monolayer state. 16,24 As demonstrated by these studies, transfer ratios are an effective means to coarsely characterize the quality of an LBdeposited SLB and can be used to efficiently optimize transfer conditions for the deposition of the highest-quality SLB for a specific composition. In the next section, these findings are corroborated using AFM topography scans to characterize the quality of the SLB on the nano- to microscopic scale. Evaluation of SLB Quality via AFM Imaging. AFM topographs enable the direct visualization of membrane morphology and nanoscopic topological defects, which are not resolvable using optical-based imaging methods. AFM measurements confirmed the earlier findings of transfer ratio measurements on the effect of deposition parameters, such as the lipid phase state, surface roughness, surface chemistry, and interleaflet couplings on SLB quality. Composition and Substrate Roughness on the Quality of the Inner Monolayer. First, the quality of LB-deposited inner monolayers was determined for pure and mixed lipids at a widely used dipping speed of 1 mm/min. The monolayers were examined using AFM to reveal their morphology and defects to provide guidance on how the quality of the inner monolayer impacts the final SLB. The surface pressure of the LB deposition for each system was chosen to be where the slope of the isotherm was the steepest. Figure 7 provides some example AFM topographs. For monolayers in the gel phase, AFM images in air revealed a relatively smooth morphology for both DPPE and DPPC monolayers, with few visible defects 0.5% and 1%, respectively. Mixed monolayers of DPPC/chol showed topographical features with 3 5 Å differences in height, corresponding to coexisting ordered phases, but no topological defects greater than 2 nm in depth. High-resolution AFM topography scans of DMPC and DTPC monolayers were not obtainable because these lipids were in the fluid phase. Even in tapping mode imaging, the fluid phase regions in an SLB can be disturbed and make resolving topological defects difficult unless the defects span the monolayer (or bilayer) film. Figure 8 shows the quality of LB-deposited gel phase DPPC monolayers on various substrates. Because of mica s low roughness (RMS of 0.2 Å), DPPC monolayers deposited on mica have fewer defects compared to DPPC monolayers on silicon wafers (RMS of 3 4 Å) or glass slides (RMS = 8 10 Å). The number of defects increases with the roughness of the substrate, consistent with the TR measurements for gel-phase inner monolayers (Table 1). Gel-phase lipids provide a more uniform and stable base monolayer for SLB formation. In general, the higher the quality of the inner leaflet, the higher the quality of the final SLB. It is also important to recall that the exchange and mixing of lipids between fluid-state leaflets are very rapid. 29,30 Deposition in the gel phase and temperature control can be used to minimize and control the kinetics of leaflet mixing. Similarly, delamination of J

11 Figure 7. Representative 10 μm 10 μm AFM topograph acquired under ambient conditions of four monolayers deposited using the LB method at a dipping speed of 1 mm/min on mica(0001). The DPPE and DPPC monolayers were deposited at a surface pressure of 45 mn/ m. The mixed 92:8 and 80:20 DPPC chol monolayers were deposited at a surface pressure of 30 mn/m. the inner monolayer during deposition of the outer layer can also occur if the physisorption to the substrate and lateral film cohesion are not sufficiently strong or if the pressure during the deposition of the outer monolayer is too low. Comparison of Symmetric and Asymmetric SLBs. As previously mentioned, drying of the inner leaflet and interleaflet coupling between the inner and outer monolayer in the SLB can affect the quality of the outer monolayer transfer. 24,127 The studies described below quantify the overall SLB quality by characterizing a series of symmetric and asymmetric SLB compositions. To minimize the parameter space, two gel-phase compositions were studied, DPPC and 92:8 DPPC cholesterol, which were also scanned as monolayers (Figure 7). The symmetric SLBs were prepared using LB LB deposition at 30 mn/m and a dipping speed of 1 mm/min for both the inner and outer monolayers with the same composition. These symmetric SLBs were compared to asymmetric SLBs, where the outer monolayer was deposited on a nearly defect-free DPPE inner monolayer. The left column of Figure 9 shows AFM topographs of symmetric DPPC/DPPC and asymmetric DPPC/DPPE SLBs. Both exhibit relatively smooth morphology. Small and randomly distributed defects are visible in the symmetric DPPC/DPPC systems, while lower-density but larger defects are seen in the asymmetric DPPC/DPPE SLBs. Even fewer defects are obtained when DPPC is deposited at 45 mn/m. To make comparisons, the outer monolayer was deposited at 30 mn/m in all cases. In the right column, both symmetric DPPC:chol DPPC:chol and asymmetric DPPC:chol DPPE SLBs exhibit higher defect densities compared to the DPPC systems. The dark regions correspond to area without outer leaflet, which expose the underlying monolayer. Defect diameters in symmetric DPPC:chol DPPC:chol layers ranged from 0.2 to 1.0 μm and covered about 16% of the SLB. Defects were much smaller in asymmetric DPPC:chol DPPE layers, 0.2 μm, and constituted only 2% of the SLB. These observations clearly demonstrate the importance of a strongly physisorbed, defectfree inner leaflet. Because of the smaller amount of water of hydration, gel-phase PE lipids, e.g., DPPE, are more strongly physisorbed to mica than PC lipids, which have about twice the amount of water of hydration per lipid headgroup. 14 Consistently, we find DPPE to be superior as a base monolayer in almost all of our studies for yielding SLBs with fewer defects. Moreover, as demonstrated in numerous measurements of bilayer interactions, the exchange between the inner and outer gel-phase leaflets is minimal on mica, enabling the interaction and properties of the outer leaflet composition membranes to be precisely determined. 14,87 89,93 Impact of Surface Pressure Area per Molecule on Overall SLB Quality. As mentioned previously, the surface pressure during deposition can be used to select the desired lipid packing (area per molecule) or phase state of the deposited monolayer and greatly influences the quality of the resulting SLB. Although TR measurements are very helpful, AFM topography scans can reveal the relationship between the surface pressure and size of membrane defects in the deposited monolayer or SLB. As a demonstration, the pressure area isotherm of 92:8 DPPC cholesterol at 25 C and AFM scans of the asymmetric SLB composed of 92:8 DPPC cholesterol deposited on DPPE at various surface pressures are shown in Figure 10. The SLB deposited at 30 mn/m shows the smallest defect size and lowest defect density. At 40 mn/m, the higher defect density and larger defect are attributed to the lower Figure 8. Representative 10 μm 10 μm AFM topograph acquired under ambient conditions of DPPC monolayers deposited by LB at a surface pressure of 45 mn/m on surfaces of (A) mica, (B) a silicon wafer, and (C) a glass slide. K

12 Figure 9. Characteristic 10 μm 10 μm AFM topographs of DPPC and 92:8 DPPC cholesterol deposited as symmetric (top row) or asymmetric (on DPPE monolayer, bottom row) SLBs on mica. The number in the top left corner of each image is the average defect density. The defect density was calculated to range from 10 to 20 AFM images per sample and from 2 to 4 samples per deposition condition. Figure 10. Pressure area isotherm of 92:8 DPPC cholesterol at 25 C. Insets are representative 10 μm 10 μm AFM topographs of asymmetric 92:8 DPPC cholesterol/dppe SLBs on mica with the outer monolayer deposited at surface pressures of 20, 30, and 40 mn/ m, respectively. stability of the monolayer at this surface pressure, where the slope of the Π A curve is beginning to decrease. At the lower surface pressure of 20 mn/m, again a larger defect size and density occur. One might hypothesize that the greater defect density at 20 mn/m compared to that at 30 mn/m is due to the condensation of the deposited film and a potential shift to a more equilibrated state. However, a careful GIXD of pure DPPC membranes LB LS deposited as a function of surface pressure on quartz demonstrated that gel-phase SLBs, for the most part, tracked and maintained the conditions under which they were deposited. 16,24 Subtle changes were observed between monolayers and bilayers because of coupling between the leaflets, but the packing and structure of the SLB followed the deposition pressure consistently. Impact of Cholesterol in Altering the Phase State and SLB Quality. Cholesterol, a specific type of sterol, is known to alter the packing or phase of 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) monolayers. The transition between the gel and liquid-ordered phase occurs at 10 mol % cholesterol. 128 In order to study the effect of cholesterol and phase state on the quality of LB deposited SLBs, binary mixtures composed of DPPC with various cholesterol concentrations were deposited on a robust, nearly defect-free DPPE inner monolayer (Figure 11). As the amount of cholesterol increases from 0 to 20 mol %, the mixed monolayer gradually transitions from a pure gel phase to a state of coexisting gel and L 0 phases. 128 AFM scans showed that as the gel-phase monolayer became more fluid, more defects were observed. Similar effects of increasing defect density with higher fluidity have been observed with other lipids and mixtures, and thus LB and LS deposition are typically performed with the lipid mixture in the gel- or solidphase state. Another advantage of deposition with gel-phase systems is a slower equilibration with the subphase (lipid dissolution into the subphase). Presaturating the working solution with lipids in the outer leaflets minimizes desorption for longer time experiments. Recently, non-lb methods for fabricating SLBs with high cholesterol concentrations have been described. The use of fusogenic peptides as well as solvent exchange methods has been shown to yield much higher quality SLBs compared to standard VF methods. 49,58 Preparation Technique and Quality of SLBs. Recent studies of biomimetic raft membranes typically involve mixtures L

13 Figure 11. AFM topography images (10 μm 10 μm) showing the defect density as a function of cholesterol concentration in a DPPC outer leaflet at 25 C. The outer monolayers were deposited on a nearly defect-free DPPE inner monolayer on mica. The uniformity in the size of the membrane defects is noteworthy. of high-melting-temperature lipids (saturated acyl chains or sphingomyelin), low-melting-temperature lipids (unsaturated acyl chains), and sterols. Depending on the ratio of the components and the temperature, the mixture could exist in the gel (L 0 ), liquid-disordered (L d ), or multiple phases (i.e., coexistence between L 0 /L d phases) which mimic the liquid liquid immiscibility region thought to exist in cellular membranes. One frequently studied system is 1:1:1 DOPC DPPC cholesterol, which has coexisting L 0 L d phases. 129 Using this system, the impact of the preparation method on the quality of the deposited SLB was investigated. We did not optimize conditions for studying phase separation. In the first case, we compared asymmetric bilayers where the first or inner monolayer was LB-deposited DPPE (45 mn/m) and the outer monolayer leaflet was deposited by LB, LS, or VF. AFM scans of the resulting SLBs clearly demonstrated that LB and LS yield more well-packed, complete membranes (Figure 1E). The outer layer of fluid-phase 1:1:1 DOPC DPPC cholesterol was LB deposited at a surface pressure of 30 mn/m and a dipping speed of 4 mm/min, LS deposited at a surface pressure of 30 mn/m, or formed by VF with 45 min of incubation using a 1 mg/ml SUV solution. In all cases, AFM scans of the resulting SLBs showed nanoscopic monolayer defects down to the inner DPPE monolayer (Figure 1E). However, the distribution/density and the uniformity of the defects varied depending on the deposition technique. SLBs with an LB-deposited outer layer had the fewest defects. The quantity of defects increased when LS was used. We hypothesize that small lateral defects form either during the deposition process or from a small condensation of the lipids when contacted with the DPPE layer. Note that for the LS technique the alignment of the substrate parallel to the monolayer (minimizing monolayer film displacement) is critical to obtaining a high-quality transfer. In the case of a VF-deposited outer layer, the quality of the SLB was much worse. Even though LB and LS yield betterquality SLBs, a majority of studies use vesicle fusion as the preparation technique. Qualitative observation with FM showed that the VF-deposited membranes were less homogeneous, but the resolution of FM measurements is insufficient for an accurate assessment of SLB quality in most cases. 126 Similarly, care must be taken in the use of QCM to establish the quality of the SLB. Adsorbed, partially fused vesicles may be present. 113 In general, SLBs formed by VF exhibit more defect densities unless great attention to detail is taken in optimizing the deposition conditions. For example, Figure 2D F displays AFM topographs of SLBs of 2:2:1 DOPC DPPC cholesterol formed by VF. Still, VF is the most straightforward method of making SLBs and the only method that can be used to perform backfilling for membrane lithography (filling up defects or areas with no bilayer or only a monolayer on the surface). 130 Figure 12 provides an Figure 12. Squares (200 μm 200 μm) of DMPC SLB enclosed by BSA created using a UV photolitography technique. The BSA was backfilled after the UV patterning of the SLB. example of UV photolithography to create a membrane pattern of DMPC squares enclosed by backfilled bovine serum albumin (BSA). Yee et al. provide details for how to UV pattern SLBs. 131,132 Additional advantages of VF methods over LB include the ability to deliver/insert polypeptides 39 or membrane proteins into SLBs and the practical ease of depositing lowmelting-point, fluid-phase lipids. Long-Term SLB Storage and Tricks of the Trade When Working with Unsaturated Lipids. Two final notes to consider are (1) the long-term storage of SLBs and (2) the potential of unsaturated lipids to oxidize. First, of course, the SLB must remain hydrated. Any exposure to air will cause the delamination of the outer bilayer leaflet. Although the solubility of lipids in water is negligibly small (<10 10 M), SLBs will equilibrate with the solution phase. 133 The desorption of lipids from the SLB is usually not a problem with vesicle fusion and solvent spreading techniques as some residual lipid usually remains in solution. In the case of LB-deposited SLBs, a small amount of lipid can be added to saturate the solution phase. Second, the oxidation of unsaturated lipids can be minimized by limiting exposure to air/oxygen by working in an inert gas such M

14 as argon or nitrogen. 134,135 This can be achieved by purchasing unsaturated lipids in chloroform in small-quantity ampules. For example, we purchase 5 mg/1 ml ampules instead of a single 25 mg/4 ml ampule for a modest fee from Avanti Polar Lipids (Alabaster, AL). Once an ampule is open, we immediately prepare the desired solution mixtures. Subsequently, LB monolayers are spread, compressed, and deposited within 30 min. We have not observed any difference in the SLB quality of LB-deposited membranes under these conditions compared to depositions in a glovebox under argon or nitrogen gas. 87,136 However, the sensitivity of specific compositions to oxidation must be carefully considered. Keller and co-workers have demonstrated substantial changes in the miscibility transitions of lipid mixtures containing unsaturated lipids and in surface pressure isotherms with as little as 1 h of air exposure. 134,137 CONCLUSIONS The solid-supported bilayer is a simple and robust system used extensively to mimic biological membranes and their properties such as the lateral topography, lipid mixing, phase state, and dynamics/diffusion. It also serves as a base system for studies with proteins and other molecules of interest using various complementary characterization techniques. This invited instructional review discussed different preparation techniques for creating SLBs with an emphasis on parameters and conditions that yield the highest-quality bilayer. Transfer ratios and membrane topological defects, which span the outer monolayer or in some cases penetrate the inner monolayer, were used to characterize the quality and completeness of the formed SLB. Optimal deposition parameters such as the surface pressure, lipid phase state, inner leaflet selection, and substrate roughness were described in detail to aid in creating the bestquality SLB for the desired study. Comparisons of SLBs prepared via different methods were performed qualitatively by fluorescence microscopy and quantitatively by transfer ratio measurements and high-resolution AFM topography scans. In general, SLBs prepared by LB LB and LB LS techniques have fewer topological defects than those formed by vesicle fusion. Residual vesicles and lipid from the fusion process may also be undesirable for some characterization or subsequent measurement techniques. In addition, LB and LS deposition allow the surface pressure and molecular packing area of the SLB to be controlled. The optimal deposition parameters for SLBs presented here are also relevant for polymer-cushioned SLBs for trans-membrane protein studies. AUTHOR INFORMATION Corresponding Author * tlkuhl@ucdavis.edu. ORCID James Kurniawan: Gang-yu Liu: Tonya L. Kuhl: Notes The authors declare no competing financial interest. Biographies James Kurniawan received a B.S. degree in chemical engineering from the University of Washington Seattle in June 2010 and M.S. and Ph.D. degrees in chemical engineering from the University of California Davis. His graduate work focused on deducing the effect of cholesterol in solid-supported biomimetic membranes using a highly sensitive surface probing technique under the advisement of Tonya L. Kuhl. James currently works at Intel Corporation, Hillsboro, Oregon. Joaõ Francisco Ventrici de Souza received his bachelor s and master s degrees in chemistry from Universidade de Sao Paulo, Brazil and his Ph.D. from the University of California Davis. While at UC Davis, he was coadvised by Tonya L. Kuhl and Gang-yu Liu, with a primary focus on using scanning probe microscopy as a tool for the nanoengineering of 3D structures and the characterization of organic thin films. Joaõ is currently a process engineer at Intel Corporation, Hillsboro, Oregon. Amanda T. Dang received her B.S. degree in materials science and engineering from the University of Washington Seattle in June She is currently pursuing a doctoral degree in materials science and N

15 engineering with a designated emphasis in biotechnology at the University of California Davis under the advisement of Tonya L. Kuhl. Her graduate research focuses on characterizing nanolipoprotein particle interactions with supported lipid bilayers. Gang-yu Liu received her B.S. from Peking University (Beijing, China) and her Ph.D. in chemistry from Princeton University. She then received a Miller Research Fellowship and did her postdoctoral research at UC Berkeley and Lawrence Berkeley National Laboratory. In 1994, she joined the faculty of the Department of Chemistry, Wayne State University and then become a faculty member at UC Davis in Currently, she is a professor of chemistry and head of the biophysics graduate group at UC Davis. Her research focuses on the development of advanced nanotechnology and high-resolution imaging and their applications in 3D nanoprinting, biomaterials science, and biochemistry. Tonya L. Kuhl received her B.S. from the University of Arizona and her Ph.D. in chemical engineering from the University of California Santa Barbara. After a stay as a postdoctoral research associate at the UCSB Materials Research Laboratory, she joined the faculty of Chemical Engineering and Materials Science at UC Davis in She is currently a professor and vice chair of the Department of Chemical Engineering and a faculty member of the Department of Biomedical Engineering and Biophysics Graduate Group. Her research focuses on the development and application of small-angle scattering techniques and interaction force measurements of interfacial thin films and soft condensed matter. ACKNOWLEDGMENTS This work was primarily supported by the NSF chemistry division through grants CHE and CHE , the Gordon and Betty Moore Foundation, and the Brazilian National Council for Scientific and Technological Development. 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