Doğangün et al. Supporting Information for. Hydrogen Bond Networks Near Supported Lipid Bilayers from Vibrational Sum

Transcription

1 Supporting Information for Page S1 Hydrogen Bond Networks Near Supported Lipid Bilayers from Vibrational Sum Frequency Generation Experiments and Atomistic Simulations Merve Dogangun, a,# Paul E. Ohno, a,# Dongyue Liang, b,# Alicia C. McGeachy, a Ariana Gray Bé, a Naomi Dalchand, a Tianzhe Li, a Qiang Cui, b,c* and Franz M. Geiger a,* a Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60660; b Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA; c Department of Chemistry, Boston University, 590 Commonwealth Ave., Boston, MA 02215, USA I. Sum Frequency Generation (SFG) Spectrometer. A. Experimental Setup. In our experiments, we utilize a Ti:Sapphire laser system (Spectra Physics Solstice) producing 795 nm pulses with an energy of 3 mj/pulse, 1 khz repetition rate and 120 femtosecond pulse duration. The optical set up is shown in Figure S1. The 795 nm beam passes through a 90/10 beam splitter (CVI, BS P) and 90% of the beam is used to pump a TOPAS tunable optical parametric amplifier (TOPAS-C, Light Conversion) with a difference frequency generation (DFG) stage to generate a broadband tunable IR beam. The TOPAS output passes through a longpass IR filter to eliminate the signal and idler beams, and the IR beam is focused onto the sample using a BaF 2 IR focusing lens with 200 mm focal length (ISP Optics, BF-PX ). The remaining 10% of the 795 nm beam is used as a visible upconverter beam, which passes through a variable density filter (ThorLabs, NDC-50C-4), and a home-built delay stage with two 3 mm thick gold mirrors (CVI, PW1-1025C) to achieve temporal overlap with the IR beam at the sample. The visible beam then passes through a narrow

2 Page S2 band-pass filter (Andover Corp. 795 nm, 1.0 nm bandwidth), to obtain a spectral resolution of ca. 10 cm -1, and an achromatic half-waveplate (Karl Lambrecht Corp., MWPAA ) for polarization rotation. The visible beam is then focused on the sample stage using an achromatic lens with 75 mm focal length (Edmund Optics, ). The incident IR beam and the 795 nm beam are directed towards the optical substrate mounted on an xyz-sample stage at 60 and 45 from the surface normal, respectively. The optical substrate is a 1 diameter and 3 mm thick CaF 2 window through which the beams pass prior to incidence upon the CaF 2 /water interface. Due to refraction, the beams are incident at the substrate/water interface at approximately 38 and 30 for the IR and visible, respectively. At the sample, the incident visible pulse energy is kept at 3.0 µj and the IR pulses are typically 8-15 µj in the O-H and µj in the C-H region. The output SFG beam is recollimated using an achromatic lens with 150 mm focal length (Edmund Optics, ), and passed through a polarizer (ThorLabs, Glan polarizer, GL15) and a half-wave plate (Karl Lambrecht Corp., MWPAA ) to control the SFG polarization for optimizing signal collection at the detector. The SFG beam then passes through a 700 nm shortpass filter (Edmund Optics, ), a plano-convex focusing lens, and a notch filter (Kaiser Optical Systems, Inc., holographic notch-plus filter, ) to remove excess 795 nm light and other light resulting from other nonlinear optical processes. A 0.5 m spectrograph (Acton SP-2558 Imaging Spectrograph and Monochromator) coupled to a liquid nitrogen cooled charged coupled device (CCD) camera (Roper Scientific, pixels) is used to detect the SFG signal. To direct beams, gold mirrors (Edmund Optics, 45606) are used in the incident IR and visible beam paths and silver mirrors (Edmund Optics, 49194) are used in the output beam paths. The set-up is enclosed with a Plexiglas box and is purged with N 2 to avoid water absorption bands.

3 Page S3 Figure S1. Schematic layout of the broadband laser setup utilized to study vibrational transitions in the C-H and O-H stretching regions. B. Spectral Acquisition and Analysis. All the SFG experiments reported here were carried out in the internal reflection geometry and the ssp polarization combination to probe the components of the vibrational transition dipole moments that are oriented along the surface normal. 1-2 SFG spectra were recorded at the supported lipid bilayer/ aqueous interface with an integration time of 1 to 4 min with an average of 3 acquisitions. Spectra were recorded sequentially using an automated Python script to adjust the IR center wavelengths ( nm) and spectrograph center wavelength ( nm) to cover the frequency range of interest ( cm -1 ).

4 Page S4 Following Esenturk and Walker, 3 we recorded each spectrum using nine different center IR wavelengths to cover the relevant frequency range of interest. Each SFG spectrum was background-corrected to account for the optical scattering of the 795 nm upconverter beam, and calibrated to the 2850 cm -1 and 3060 cm -1 (NIST 1921b) peaks of a 70 µm thick standard polystyrene film (International Crystal Laboratories). Each spectrum was normalized to the incident IR energy profile by recording a nonresonant spectrum from a gold film on CaF 2, following the procedure detailed by Liljeblad and Tyrode. 4 A gold spectrum is recorded (Figure S2a) for each IR center wavelength used in recording the sample spectrum (Figure S2b). The individual gold spectra are then smoothed with a Gaussian filter and truncated at a cutoff value of 5% of their maxima (Figure S2c). The individual sample spectra are truncated at the same positions as their gold counterparts (Figure S2d). This truncation prevents adding into the final spectrum noise generated at the detector from spectral regions where there was little to no incident IR energy present. After truncation, the individual gold (Figure S2e) and sample (Figure S2f) spectra are summed into the total, composite spectra. The final normalization occurs when the summed and truncated sample spectrum is divided by the normalized, summed and truncated gold spectrum.

5 Page S5 40x10 3 a 800 b I SFG [c.p.s.] x10 3 c d I SFG [c.p.s.] x e f I SFG [c.p.s.] Wavenumber [cm -1 ] Wavenumber [cm -1 ] Figure S2. Gold normalization example a) Individual non-resonant gold spectra b) individual sample spectra c) gold spectra smoothed and truncated at 5% of their maximum value d) sample spectra truncated at positions determined by gold counterparts e) summed gold spectrum f) summed sample spectrum. Note the removal of baseline noise between (b) and (d) prior to the final summation.

6 Page S6 II. Fluorescence Recovery after Photobleaching (FRAP) Experimental Procedure and Analysis. FRAP measurements and sample preparation were carried out in a manner consistent with our previous approach. 5 FRAP measurements were carried out using a Leica Spinning Disk Microscope (Leica DMI6000 inverted microscope equipped with a Yokogawa CSU-X1 Spinning Disk module, Photometrics Evolve Delta512 camera) with a 63x oil immersion objective (HC PL APO 1.4NA). Here, we use an ilas 6 attachment mounted onto the microscope (Roper Scientific, 401 nm 50 mw; 100% of laser power) for photobleaching. Images were collected with the Green ET525/50M emission filter and 488 nm, 50 mw laser at 10-15% power. Metamorph was used for data collection and ImageJ was used for data processing. The simfrap plugin for ImageJ was used to extract lateral diffusion coefficients. 7 Supported lipid bilayers were formed as described in Section IIA, using a homebuilt Teflon flow cell, 1-inch diameter calcium fluoride window (ISP Optics, CF-W-25-1) marked on the edge with marker (to facilitate alignment). Specifically, the calcium fluoride window was clamped onto the Teflon flow cell to create a leak-tight seal. The cell was equilibrated with 0.1 M NaCl, 0.01 M Tris buffer (ph 7.4). A vesicle containing solution (0.5 mg/ml) was then introduced into the flow cell and an SLB was allowed to form via the vesicle fusion method for at least 15 minutes. Immediately after rinsing with either buffer and/or Millipore water ph adjusted to 7.4, the window was removed and seated on a glass coverslip with a thin layer of silicone grease securing the perimeter of the window to the glass coverslip. Additionally, a small reservoir of either buffer or Millipore water was used as a hydration layer between the window and coverslip to match the conditions of the last rinse solution. FRAP measurements were carried out at this point for SLBs formed from DMPC, 9:1 DMPC/DMPG, and 8:2

7 Page S7 DMPC/DMPG (Figure S3). The errors associated with the diffusion coefficients reported in the main text are the standard errors determined by dividing the standard deviation of the diffusion coefficients for each condition by the square root of the number of replicates. All the individual diffusion coefficients, along with their associated errors reported from simfrap, are presented in tabular form (Table S1). Fitting Bounds Real Simulated 0.1 M NaCl ph-adjusted Millipore H 2 O

8 Page S8 Figure S3. Representative simfrap traces for DMPC (top row), 9:1 DMPC/DMPG (middle row) and 8:2 DMPC/DMPG after rinsing with 0.1 M NaCl buffer (left column) and ph-adjusted Millipore water (right column). While diffusion coefficients associated with supported lipid bilayers formed from zwitterionic lipids on silicon oxide seems to be independent of ionic strength, SLBs formed on alumina, 8 some glasses, 9 and mica 9 have increased diffusion coefficients with increasing ionic strength. This observed correlation between the solid support and the observed diffusion coefficient was attributed to the thicker layer of interfacial water associated with aluminum oxide. To the best of our knowledge, there are no reports that describe the diffusion of SLBs on calcium fluoride as a function of ionic strength. Here we find that reducing the ionic strength from 0.1 M to <0.1 mm (ph adjusted Millipore) above CaF 2 -supported DMPC and 9:1 DMPC/DPMG bilayers results in a decrease in the observed diffusion coefficient. In fact, this nearly one order of magnitude reduction in diffusion coefficient is consistent with the reduction in diffusion coefficient observed with glass- and mica- supported lipid bilayers formed from zwitterionic lipids around their transition temperature when the ionic strength is reduced from 0.1 M NaCl to zero salt. 9

9 Page S9 Table S1. Individual diffusion coefficients and errors as determined by simfrap plugin for SLBs after rinsing with 0.1 M NaCl buffer or ph-adjusted Millipore water. DMPC 9:1 DMPC/DMPG 8:2 DMPC/DMPG 0.1 M NaCl H 2 O 0.1 M NaCl H 2 O H 2 O ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.07 III. Sample Preparation A. Flow Cell. The details of the flow cell used in our experiments have been previously described in detail The SFG spectra were collected using a home-built Teflon flow-cell with a volume of approximately 3.5 ml. The Teflon cell was sealed off with a Viton O-ring (Chemglass) and an optical window (1 diameter) using an aluminum face plate attached to Teflon. The sample solutions were flowed through the reservoir across the interface with a variable flow rate controller at 1.5 ml/min. The CaF 2 window was sonicated in methanol (Fisher Scientific, HPLC- Grade) for 30 min, rinsed with Millipore water (18 Ω cm resistivity; Millipore), and dried under a stream of N 2. The flow cell and PTFE tubing were sonicated in methanol for 15 min, rinsed with Millipore water and dried with N 2. The window, flow cell and O-ring were plasma cleaned (Harrick Plasma Cleaner, PDC-32G, 18W) for 10 min, immediately before use.

10 B. Bilayer Formation. Page S10 Details of the vesicle preparation have been reported previously ,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1-rac-glycerol) (DMPG) were purchased from Avanti Polar Lipids and used without further purification. Pure DMPC lipids, a mixture containing 9:1 DMPC/DMPG and 8:2 DMPC/DMPG lipids were dried under a stream of N 2 and rehydrated in a 10 mm Tris buffer solution at ph 7.4, with 100 mm NaCl and 5 mm CaCl 2 2H 2 O. The lipids were extruded using a mini-extruder kit (Avanti Polar Lipids) and a 0.05 µm polycarbonate membrane filter to form small unilamellar vesicles. 12 The extruded lipids were injected into the flow-cell and lipid bilayers were formed on a CaF 2 surface via vesicle fusion method All bilayers were rinsed with 20 ml of calcium-free buffer solution (100 mm NaCl, 10 m M Tris, ph 7.4) to ensure removal of excess vesicles at the interface. The bilayers were then rinsed with 20 ml of low ionic strength solution (no buffer) at ph 7.4 and spectra were recorded. Then 20 ml of aqueous solution with 100 mm NaCl (no buffer) at ph 7.4 was introduced and SFG spectra were recorded. Before the preparation of low and high ionic strength aqueous solutions, Millipore water was left overnight to equilibrate with atmospheric CO 2. The solution ph was measured for each salt concentration and the ph was adjusted to 7.4 with minimal NaOH and HCl before the solutions were flowed across the interface. For low ionic strength solution, there is no additional salt. For the high ionic strength condition, we used 0.1 M NaCl. IV. SFG Spectra A. Substrate Choice We used CaF 2 windows (ISP Optics, #CF-W-25-3) as solid substrates for the experiments reported here due to optical adsorption from the fused SiO 2 substrate (ISP Optics, #QI-W-25-3)

11 Page S11 in the O-H and C-H stretching regions. Despite the differences optical absorption seen below 2900 cm -1, the frequencies corresponding to the vibrational features in the C-H stretching region shown in Figure 1 are comparable to the ones we observe for supported lipid bilayers formed on fused silica substrates. Figure S4 shows similar spectral shapes from SLBs made from a 9:1 mixture of DMPC/DMPG lipids on fused SiO 2 and CaF 2 windows. Yet on CaF 2, the water interference to the C-H stretching region is clearer as the system was not tuned to the O-H region when experiments were carried out on the fused SiO 2 substrates ISFG [a.u.] Wavenumber [cm -1 ] 3200 Fig S4. ssp-polarized SFG Spectra of SLBs made from a 9:1 mixture of DMPC/DMPG on a fused SiO 2 window (brown) and CaF 2 window (green).

12 B. D 2 O Exchange Page S12 Figure S5 shows that switching between H 2 O and D 2 O in the same flow cell, eliminates the O-H signal while retaining the C-H oscillators I SFG [a.u.] Wavenumber [cm -1 ] Fig. S5. ssp-polarized SFG spectra of an SLB made from a 9:1 mixture of DMPC/DMPG lipids in 100 mm NaCl on a CaF 2 window in H 2 O, ph 7.4 (green) and in D 2 O, pd ~7-8 (black) at 21 ± 2 ºC.

13 V. CaF 2 Dissolution and Controlled Inhibition by Lipid Bilayer. Page S13 A. CaF 2 /H 2 O Spectra (liquid) A I SFG [a.u.] Wavenumber [cm -1 ] B Figure S6: ssp-polarized SFG spectrum of neat water adjusted to ph 7.4 with a flow rate of 1.5 ml/min (blue) and with no flow (red) (A) for CaF 2 /water and (B) SLB/water interface (unnormalized).

14 B. CaF 2 /H 2 O Spectra (vapor) Page S14 The CaF 2 /H 2 O(g) interface was probed using a home-built relative humidity (RH)-controlled flow system, shown in Figure S7, which has been described in detail previously. 15 Briefly, ultrahigh purity helium gas was passed through separate dry and humidified wet air flow lines and subsequently sent across a clean CaF 2 optical window, which was sealed with a Viton O-ring to a home-built Teflon flow-cell. Analogous to Section IIA, optical windows were rinsed and sonicated with water and methanol and then plasma cleaned prior to measurement. Helium was sent at a flow rate of 0.5 SLPM (standard liter per minute) through the dry air path (<0.5% RH) and 0.2 SLPM through the wet (>80% RH) air path consisting of a bubbler with roughly 10 ml of Millipore water. The helium flow rates were regulated using electronic mass flow controllers (MFCs) (Alicat Scientific), and the RH was measured using an RH meter (Omega Engineering, RH-USB) attached to the flow-cell outlet. The %RH took approximately 2 min to stabilize upon switching between dry and wet air flow lines. Figure S7. a) Schematic of home-built relative humidity (RH)-controlled flow system, b) Plot of %RH for a representative cycle between dry and wet conditions.

15 VI. Atomistic Simulations Page S15 We used structure and input files generated by CHARMM-GUI input generator for equilibration and production runs. For equilibration, CHARMM-GUI provides input for a six-step equilibration process. Throughout the six-step equilibration, Langevin dynamics was applied, with force switching applied to van der Waals interactions with a cutoff of 1.2 nm. Particlemesh-Ewald is used for electrostatic interactions, with a real-space cutoff of 1.2 nm and grid sizes consistent with production runs. The temperature for each run was K. The ensemble, timestep, equilibration time, and restraints applied are listed in Table S2. Table S2. Detailed information of the equilibration protocol for MD simulations. Step Ensemble 1 Timestep Time Planar 2 Dihedral 3 1 NVT 1 fs 25 ps NVT 1 fs 25 ps NPAT 1 fs 25 ps NPAT 2 fs 100 ps NPAT 2 fs 100 ps NPAT 2 fs 100 ps NPAT ensemble stands for constant normal pressure, lateral surface area and temperature. 2 Scaled force constants in the units of kcal/mol of planar harmonic restraints applied to lipid head groups of the upper/lower layer with centers of z = ± 17 Å. 3 Force constants for dihedral restraints to keep the C2 chirality of each lipid in kcal/mol.

16 Page S16 Figure S8. Mass density distribution of lipid molecules (shown in solid lines) and water (shown in dashed lines) from atomistic MD simulations. From the distribution, the interface is located at z ~ 20 Å.

17 Page S17 Figure S9. Snapshots from MD simulation of a solvated DMPC lipid bilayer system. The snapshot on the right illustrates the water molecules that penetrate below the phosphate-water interface and engage in hydrogen bonding interactions with the glycerol oxygen in the lipid (z~10 Å). Color representations: water oxygen red, lipid nitrogen blue, lipid phosphorus tan, carbon green, hydrogen white.

18 Page S18 Figure S10. Left: distribution of orientations of the phosphate-nitrogen vector of the DMPC head group from the atomistic MD simulation of a solvated DMPC bilayer. Right: distribution of water dipole angle from atomistic MD simulations. Φ is defined as the angle between the phosphate-nitrogen vector and membrane normal. As in Figure 3 in the main text, the left distribution is divided by the Jacobian factor sinφ. The distribution of Φ is broad, which leads to a broad distribution of water orientation near the lipid-water interface.

19 VII. Supported Lipid Bilayers at Low and High Ionic Strength Page S19 Figure S11 demonstrates the triplicates of each bilayer formed from pure DMPC lipids and 9:1 and 8:2 mixtures of lipids containing DMPC and DMPG lipids on a CaF 2 window. We observe three features from the C-H oscillators, centered around 2860, 2920 and 2970 cm -1 and two broad features from the O-H oscillators centered around 3200 and 3400 cm -1. The C-H features from the CaF 2 supported lipid bilayers are in agreement within uncertainty (± 10 cm -1 ) with the oscillators reported earlier, from fused silica supported lipid bilayers. 1.2 A 1.0 I SFG [a.u.] Wavenumber [cm -1 ]

20 Page S B I SFG [a.u.] Wavenumber [cm -1 ] 1.0 C I SFG [a.u.] Wavenumber [cm -1 ]

21 Page S D I SFG [a.u.] Wavenumber [cm -1 ] E I SFG [a.u.] Wavenumber [cm -1 ]

22 Page S F I SFG [a.u.] Wavenumber [cm -1 ] G I SFG [a.u.] Wavenumber [cm -1 ]

23 Page S H I SFG [a.u.] Wavenumber [cm -1 ] Figure S11: ssp-polarized SFG spectrum of an SLB formed from (A,B) pure DMPC (C,D,E) 9:1 mixture of DMPC/DMPG (F,G,H) 8:2 mixture of DMPC/DMPG (blue) lipids in Millipore water with no added salt (lighter lines) and with 100 mm NaCl (darker lines) at 21 ºC and ph 7.4. Lines represent the data that have been binned by over nine points in x and y between 3000 cm -1 and 3600 cm -1.

24 VIII. Additional Atomistic Calculations Page S24 Figure S12. Radial distribution functions (RDF) of water oxygens with respect to lipid nitrogen and phosphorus. N(r) is the integrated RDF. In the work of Morita and coworkers, water oxygens within 1 st /2 nd shell of corresponding RDFs are regarded as adjacent to lipid nitrogen/phosphorus. In our case, the corresponding distances are 5.95/6.95 Å.

25 Page S25 Figure S13. The orientation of NP water (water molecules close to both choline and phosphate groups) in comparison with all water from atomistic MD simulations for the DMPC system without salt. Although there is a small systematic difference, the variance of the orientation for the NP water is still large, reflecting the thermal fluctuation of the lipid/water interface (Figure S14).

26 Page S26 Figure S14. The average and standard deviation of nearest O O distance of water near the lipid bilayer/water interface from atomistic MD simulations. Lipid composition and salt concentration are shown in the legend. The z axis represents the membrane normal, and z = 0 is located at the center of bilayer. Each analysis is averaged over 1000 frames.

27 Page S27 Figure S15. Two-dimensional water orientation distribution and nearest water O O distance (average and standard deviation) for a solvated DMPC bilayer system at K after 30 ns of atomistic MD simulation. The system has otherwise identical set-up to the salt-free DMPC system discussed in the main text. Results are averaged over 1000 frames. By comparing to Figure 3 in the main text, no significant variation is observed due to the temperature difference.

28 IX. Method for Estimating Surface Potential Changes from SFG Signal Intensity Page S28 Differences. We start with the overall equation: χ "#$ = χ " + χ "#$ + "#$% e Φ 0 χ Eqn. S1 Assuming the non-resonant intensity is zero, the difference between two intensity spectra at two different potentials is: ΔI "# = I "#," I "#, χ "#$ + " " e "Φ " 0 χ χ "#$ + e Φ 0 χ Eqn. S2 where φ = arctan k κ Explicitly writing out the real and imaginary components leads to: = χ "#$," i χ "#$," χ "#$," i χ "#$," + χ " " " cos φ " Φ " 0 χ " " " sin φ " Φ " χ " " " cos φ " Φ " 0 + χ " " " sin φ " Φ " 0 + χ " cos φ Φ 0 χ " sin φ Φ χ " cos φ Φ 0 + χ " sin φ Φ 0 Eqn. S3 We then explicitly calculate the square magnitude of the low and high salt terms: = χ "#$," + χ" " " cos φ " Φ " 0 + χ " " " sin φ " Φ " 0 + χ "#$," χ " cos φ " " " Φ " 0 χ "#$," χ "#$," + χ" " " cos φ " Φ 0 χ " " " sin φ " Φ " χ " " " sin φ " Φ " 0 +

29 Page S29 χ "#$," χ " " " cos φ " Φ " 0 + χ "#$," χ " " " sin φ " Φ " 0 χ "#$," + χ" cos φ Φ 0 + χ " sin φ Φ 0 + χ "#$," χ cos φ " Φ 0 χ "#$," χ "#$," + χ" cos φ Φ 0 χ " sin φ Φ χ " sin φ Φ 0 + χ "#$," χ " cos φ Φ 0 + χ "#$," χ " sin φ Φ 0 Eqn. S4 and find the difference: = χ " + χ" " " Φ " 0 Φ 0 + χ "#$," χ " cos φ " " " Φ " 0 χ "#$," χ " sin φ " " " Φ " 0 χ "#$," χ " cos φ " " " Φ " 0 χ "#$," χ " sin φ " " " Φ " 0 cos φ Φ 0 sin φ Φ 0 + cos φ Φ 0 + sin φ Φ 0 Eqn. S5 The first term represents the square magnitude of the bulk χ (3) spectrum of water multiplied by a scalar determined by the varying ionic strengths and potentials. The latter four terms contain the second-order spectral contributions from the surface. If we assume the overall spectra are dominated by the χ (3) contribution, i.e. χ (3) *Φ >> χ (2), the first term dominates and the difference in potential can be estimated if the magnitude of the difference in I SFG is known.

30 References. Page S30 1. Esenturk, O.; Walker, R. A., Surface Structure of Strongly Associating Liquids: Vibrational Studies of Nonanols and Nonanones. Abstr. Pap. Am. Chem. S 2004, 228, U286- U Zhuang, X.; Miranda, P. B.; Kim, D.; Shen, Y. R., Mapping Molecular Orientation and Conformation at Interfaces by Surface Nonlinear Optics. Phys. Rev. B 1999, 59, Esenturk, O.; Walker, R. A., Surface Vibrational Structure at Alkane Liquid/Vapor Interfaces. J. Chem. Phys. 2006, Liljeblad, J. F. D.; Tyrode, E., Vibrational Sum Frequency Spectroscopy Studies at Solid/Liquid Interfaces: Influence of the Experimental Geometry in the Spectral Shape and Enhancement. J. Phys. Chem. C 2012, 116, Olenick, L. L.; Chase, H. M.; Fu, L.; Zhang, Y.; McGeachy, A. C.; Dogangun, M.; Walter, S. R.; Wang, H.-f.; Geiger, F. M., Single-Component Supported Lipid Bilayers Probed Using Broadband Nonlinear Optics. Phys. Chem. Chem. Phys Wu, E. S.; Jacobson, K.; Papahadjopoulos, D., Lateral Diffusion in Phospholipid Multibilayers Measured by Fluorescence Recovery after Photobleaching. Biochem. 1977, 16, Blumenthal, D.; Goldstien, L.; Edidin, M.; Gheber, L. A., Universal Approach to Frap Analysis of Arbitrary Bleaching Patterns. Sci. Rep. 2015, Jackman, J. A.; Tabaei, S. R.; Zhao, Z. L.; Yorulmaz, S.; Cho, N. J., Self-Assembly Formation of Lipid Bilayer Coatings on Bare Aluminum Oxide: Overcoming the Force of Interfacial Water. ACS Appl. Mater. Inter. 2015, 7, Harb, F. F.; Tinland, B., Effect of Ionic Strength on Dynamics of Supported Phosphatidylcholine Lipid Bilayer Revealed by Frapp and Langmuir-Blodgett Transfer Ratios. Langmuir 2013, 29, Dogangun, M.; Hang, M. N.; Troiano, J. M.; McGeachy, A. C.; Melby, E. S.; Pedersen, J. A.; Hamers, R. J.; Geiger, F. M., Alteration of Membrane Compositional Asymmetry by Licoo2 Nanosheets. ACS Nano 2015, 9, Troiano, J. M., et al., Direct Probes of 4 Nm Diameter Gold Nanoparticles Interacting with Supported Lipid Bilayers. J. Phys. Chem. C 2015, 119, Szoka, F.; Papahadjopoulos, D., Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes). Annu. Rev. Biophys. Bio. 1980, 9, Castellana, E. T.; Cremer, P. S., Solid Supported Lipid Bilayers: From Biophysical Studies to Sensor Design. Surf. Sci. Rep. 2006, 61, Schonherr, H.; Johnson, J. M.; Lenz, P.; Frank, C. W.; Boxer, S. G., Vesicle Adsorption and Lipid Bilayer Formation on Glass Studied by Atomic Force Microscopy. Langmuir 2004, 20, Shrestha, M.; Zhang, Y.; Upshur, M. A.; Liu, P.; Blair, S. L.; Wang, H.-f.; Nizkorodov, S. A.; Thomson, R. J.; Martin, S. T.; Geiger, F. M., On Surface Order and Disorder of Α-Pinene- Derived Secondary Organic Material. J. Phys. Chem. A 2015, 119,