Robust Self-Referencing Method for Chiral Sum ... - ACS Publications

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Robust Self-Referencing Method for Chiral Sum Frequency Generation Spectroscopy M. Luke McDermott and Poul B. Petersen* Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States S Supporting Information *

ABSTRACT: Chiral sum frequency generation spectroscopy (SFG) is of great interest for studying biological systems, among others. Whereas the chiral response in circular dichroism is about 0.1% of the achiral response, the chiral SFG response can be the same order of magnitude as the achiral SFG signal. However, chiral SFG is limited by the attainable signal-to-noise of the weak nonlinear signals and therefore extremely sensitive to proper alignment. We present a robust method for chiral SFG and demonstrate the use on solid−air surfaces with achiral and chiral molecules. We simultaneously measure two orthogonal polarizationseither the interference chiral SFG (±45° polarized) or the pure chiral and achiral SFGusing a waveplate and beam displacer. Both optics are placed in the detection arm and can be easily incorporated into any SFG setup. Furthermore, we employ self-referencing to calibrate alignment for each sample individually using a polarizer in the detection arm. These methods greatly increase the reliability and quality of chiral SFG measurements.

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INTRODUCTION Vibrational sum frequency generation spectroscopy (SFG) has emerged as a powerful tool for studying interfaceswhich exhibit a net orderingdue to the requirement for broken inversion symmetry.1,2 Chirality is another form of ordering that breaks inversion symmetry, and chiral SFG spectroscopy is an emerging method for studying biological molecules at interfaces.3−8 Achiral SFG spectroscopy has allowed structural characterization of a range of biologically and technologically important surfaces with chromophores at submonolayer densities. Chiral SFG has the potential to connect the structural information from traditional SFG spectra to biologically relevant information about the chiral structure of proteins and biomolecules. For example, chiral SFG was used to observe the α-helix to β-sheet transition of amyloid aggregation at the water−lipid surface in situ in real time.5 As spectroscopic techniques improve, chiral SFG has further potential in realtime experiments on fundamental questions of chiral recognition and protein conformational changes as well as nonbiological systems. Second-order nonlinear chiral spectroscopies (e.g., chiral SFG) have several key advantages over linear chiral spectroscopies. While intrinsic chirality of stereocenters dominates linear chiroptical signals, structural chirality of achiral chromophores arranged in chiral structures dominates second-order nonlinear chiroptical signals.9,10 In practice, linear chiroptical spectroscopy detects chiral signals about 0.1% as large as background linear achiral signals, whereas chiral SFG signals can be near the same order of magnitude as achiral SFG signals and can be detected without achiral background.9,10 The sensitivity to structural chirality has allowed in situ chiral SFG of protein secondary structure at surfaces.4 Recent investigations have © 2015 American Chemical Society

expanded the detection limits of chiral SFG to allow study of faster processes and more dilute and weaker chromophores.6,7 Despite these advances, chiral SFG remains challenging. The most rigorous chiral SFG method to date is the twin polarization angle approach, which was developed to make chiral SFG results more accurate.11 While improving the accuracy, the twin polarization angle approach significantly increased the complexity and amount of data that needs to be collected. Here we present a simple, cheap, and reliable method of chiral SFG that extends the detection limits of chiral SFG without complicating the experiment. The method consists of self-referencing and polarization multiplexing and can be easily adapted to most SFG spectrometers. We measure two polarizations of the SFG intensity simultaneously (i.e., polarization multiplexing)either positively and negatively interfered chiral and achiral signals or pure chiral and achiral signals. Additionally, the chiral SFG signal is calibrated for each sample (i.e., self-referencing). Below we demonstrate the improved robustness and certainty of measured chiral SFG signals. The origin of the chiral sensitivity of the SFG signal has been discussed previously.12 Briefly, in a typical SFG experiment, a nonresonant visible photon and a resonant infrared photon induce a nonlinear polarization in the sample that emits a detected photon at the sum frequency. ωSFG = ωvis + ωIR Received: August 21, 2015 Revised: August 29, 2015 Published: August 31, 2015 12417

DOI: 10.1021/acs.jpcb.5b08176 J. Phys. Chem. B 2015, 119, 12417−12423

Article

The Journal of Physical Chemistry B

Figure 1. Detection arm for polarization-multiplexed interference chiral SFG. Use of waveplate and beam displacer allows the mixing of the stronger achiral signal (blue) and weaker chiral signal (red), so that the ±45° interference chiral SFG mixtures (purple) are simultaneously detected on the CCD array.

Figure 2. Detection arm for simultaneous pure chiral and achiral SFG detection. Rotating the waveplate to 0° allows the beam displacer to spatially separate the stronger achiral signal (blue) and weaker chiral signal (red) for simultaneous detection by the CCD camera.

While previous interference chiral SFG experiments have utilized the pθp polarization combinations by polarizing the visible beams to θ, our interference chiral SFG experiments collect an intermediate SFG polarization (i.e., ±45°sp and ±45°pp), which is essential for simultaneous detection. θsp and pθp interfere the same achiral and chiral polarizations, but controlling the polarization of the SFG beam is more robust than changing the polarization of the visible beam because the incident beams at the sample are not changed. For p-polarized visible and infrared beams, measuring the emitted ±45° polarized SFG intensity produces the achiral ± chiral combinations.

The polarization that generates the output sum frequency depends on the visible and infrared incident electric fields as well as the effective second-order nonlinear susceptibility. (2) P(2) = χeff EvisE IR

Waveplates and polarizers control the polarization of each of the three beams (sum frequency, visible, and infrared) to be parallel (p) or perpendicular (s) relative to the plane of incidence of the visible and infrared beams on the sample. Polarizations are listed in descending order of photon frequency (e.g., an ssp experiment indicates s-polarized SFG, s-polarized visible, and p-polarized IR). For a surface that is symmetric under rotation about the surface normal, four of the possible SFG polarization combinationsppp, ssp, sps, and pssmeasure vibrations from achiral structures, and three combinationspsp, spp, and ppsmeasure vibrations from chiral structures. A rule of thumb is that the achiral combinations always have an odd number of p-polarized beams, while the chiral combinations have an even number of p-polarized beams. Comprehensive treatments of chiral SFG theory are available in the literature.9,10,12−16 In the C∞ point group, the three chiral combinations probe the same chiral susceptibility element, χ(2) zyx, as explained by Yan et al. using the C4 subgroup.13 Therefore, the choice between spp, psp, and pps depends only on Fresnel factors, beam angles, and optics. Here we demonstrate spp and psp chiral measurements, while pps was not attempted because the signals arising from the s polarization of the IR beam are generally weaker.17 The first chiral SFG spectra of surfaces were collected using interference to mix strong achiral and weak chiral polarizations,3 a method first developed for bulk solutions.18 Interference chiral SFG mixes a stronger achiral signal with a weaker chiral signal using a polarization between the conventional s and p. For example, when polarizing the SFG signal at an angle θ, the polarization is a trigonometric combination of s and p.

I±45 ° pp ∝ |χ±(2) |2 45 ° pp

χ±(2) 45 ° pp

=

(2) (2) χppp ± χspp

2

In other words, the stronger achiral signal heterodynes the weaker chiral signal. The difference between the +45°pp and −45°pp signals is directly proportional to the chiral signal, which means both signals need to be collected. (2) I+45 ° pp − I −45 ° pp ∝ 2|χ (2) ppp||χ spp|cos φ

where φ is the relative phase between the achiral and chiral susceptibilities. Previous interference chiral SFG experiments have collected pθp polarizations separately, rotating polarizers and waveplates between data collection.1,3,11,19,20 Since the chiral response is typically weak, slight changes in alignment and laser power can have significant effects on the observed differences between the interference signals. To overcome these challenges, we simultaneously measure the two interference polarizations so that alignment and laser power are identical between the two. The use of a calcite beam displacer to simultaneously collect multiple achiral polarizations (i.e., polarization multiplexing) has been reported.17 This previous study used a beam displacer to simultaneously collect four orthogonal SFG polarizations using three incident beams: an infrared beam polarized close to the magic angle (i.e., containing both s and p polarizations) and two separate visible beams polarized at s and p, respectively. Under the assumptions of an achiral surface (i.e., no chiral tensor elements), all four achiral polarization combinations were detected simultaneously. Our method extends polarization multiplexing to detect chiral

polarization(θ ) = cos(θ )p + sin(θ )s

The second-order susceptibility for the polarization combination θpp combines the achiral ppp with the chiral spp. (2) (2) χθ(2) = cos(θ)χppp + sin(θ)χspp pp

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DOI: 10.1021/acs.jpcb.5b08176 J. Phys. Chem. B 2015, 119, 12417−12423

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Figure 3. θpp chiral SFG of the CH stretching region of spin-coated PMMA and adsorbed ubiquitin. Interference and pure chiral SFG of (a) PMMA and (b) ubiquitin. Interference and chiral SFG comparison for (c) PMMA and (d) ubiquitin. Self-referencing of (e) PMMA and (f) ubiquitin. The achirality of PMMA and chirality of ubiquitin are clear in both the interference and pure chiral SFG. The self-referencing blocks the chiral signal.

pps. The simultaneous detection and self-referencing (described below) remove the uncertainty about whether the weak chiral signal is leak-through of the achiral signal, which can be much larger than the chiral signal.20 Figure 2 shows the pure chiral SFG detection setup. The achromatic waveplate can be removed or simply rotated to 0° (as in this study). The beam displacer then spatially separates the achiral signal and chiral signal, which are simultaneously detected on the CCD array.

SFG responses without the additional beam used in that study or assumptions about the surface’s chirality. Crucially, the additional optics of our setup are only after the sample, allowing the use of a removable polarizer, rotatable achromatic waveplate, and thick beam displacer without concern over the effect on timing, pointing, or dispersion at the sample. Figure 1 shows the detection arm of the SFG spectrometer. Emitted sum frequency from a chiral sample contains orthogonal achiral (e.g., ppp) and chiral (e.g., spp) polarizations. In the absence of the self-referencing polarizer, the achromatic waveplate rotates the polarizations 45°. The beam displacer then spatially separates the vertically and horizontally polarized beams, forming plus and minus combinations of the achiral and chiral signals. Detection by CCD (charge-coupled device) camera resolves the two interference chiral SFG signals simultaneously. For interference chiral SFG, simultaneous detection eliminates uncertainty that the differences between positively and negatively interfered combinations are caused by pointing changes or fluctuating signal strength. The value of polarization control in the SFG detection arm also applies to pure chiral SFG. In traditional pure chiral SFG spectrometers using polarizers, the achiral SFG beam is generated and then immediately discarded by the polarizer. Our method uses a beam displacer to collect the achiral and chiral signals together: ppp with spp, ssp with psp, or sps with

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EXPERIMENTAL METHODS In the SFG spectrometer of the current study, a 3 mJ beam from a Ti:sapphire amplifier (Coherent, Legend Elite Duo, 793 nm, 1 kHz, 25 fs, 38 nm fwhm) pumps an OPA (OPerA Solo, Coherent) to generate 3400 nm infrared (250 cm−1 fwhm, 30 μJ), which is filtered (germanium, 3000 nm long-pass, Spectrogon) and focused at the sample at an incident angle of 40° with a pulse energy of 5 μJ. A separate pulse from the amplifier passes through an etalon (TecOptics, 795 nm, 0.6 nm, 10 cm−1) and bandpass filter (Thorlabs, 800 nm, 10 nm fwhm) to create a narrowband pulse focused on the sample at an incident angle of 55° with a pulse energy of 5 μJ. The SFG signal reflected from the solid−air surface of a chiral sample contains orthogonally polarized chiral and achiral signals, both of which are rotated by 45° when the λ/2 waveplate (air12419

DOI: 10.1021/acs.jpcb.5b08176 J. Phys. Chem. B 2015, 119, 12417−12423

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Figure 4. θsp chiral SFG of the CH stretching region of spin-coated PMMA and adsorbed ubiquitin. Interference and pure chiral SFG of (a) PMMA and (b) ubiquitin. Interference and chiral SFG comparison for (c) PMMA and (d) ubiquitin. Self-referencing of (e) PMMA and (f) ubiquitin. The achirality of PMMA and chirality of ubiquitin are clear in both the interference and pure chiral SFG. The self-referencing blocks the chiral signal.

used in SFG spectroscopy,21 and collagen has been a common starting point for chiral SFG studies.9,22,23 Ubiquitin from bovine erythrocytes (Sigma U6253) was dissolved in ultrapure water (EMD Millipore Milli-Q Advantage A10 system, 18.2 MΩ cm at 25 °C, 5 ppb TOC), placed in contact with a CaF2 window overnight, and then rinsed with ultrapure water. Collagen from rat tail (Sigma C7661) was dissolved at 1 mg/ mL in acetic acid for 3 h and drop-cast onto a CaF2 window and used immediately. In the interference chiral SFG setup (Figure 1), the +45° and −45° interference signals are polarized horizontally and vertically by the beam displacer and reflect differently off the detector grating. Self-referencing (discussed in detail below) calibrates for the grating reflection efficiency and ensures that the difference between the +45° and −45° SFG signals represents chirality. Briefly, the polarizer blocks the chiral signal, allowing only the achiral signal to pass through to the detector. The achiral signal is then rotated 45° by the waveplate and split by the beam displacer into two beams of equal intensity. The two beams do not reflect equally off the grating because of their orthogonal polarizations. Dividing the intensity of the two beams reveals a scalar grating efficiency factor (for more details see the Supporting Information). The polarizer is removed, and the grating efficiency factor can be used to calibrate interference and pure chiral SFG signals. A post collar

spaced, Eksma 467-4205) is set to 22.5° (Figure 1). To increase reproducibility, the waveplate was placed in a motorized rotation mount (Newport NewStep NSR1). The use of an achromatic waveplate is essential because of the breadth of SFG wavelengths in broadband SFG detection. The birefringent calcite beam displacer (Thorlabs BD27) projects half of each of the 45° rotated chiral and achiral signals onto spatially separated horizontally and vertically polarized beam paths directed into a monochromator (Acton SP-2500i, Princeton Instruments, 1800 grooves/mm blazed at 500 mm) and collected on a liquid nitrogen-cooled CCD array (Spec-10, Princeton Instruments, 1340 × 400 pixels). Sample signals are normalized to the infrared envelope obtained from nonresonant gold SFG signals in the ppp polarization. An octadecyltrichlorosilane (OTS) self-assembled monolayer (SAM) and spin-coated poly(methyl methacrylate) (PMMA) film were used as model achiral samples because of their common use in SFG spectroscopy. OTS (Sigma-Aldrich) was prepared at 2 mM in hexanes and deposited on SiO2-coated CaF2 windows. The CaF2 windows (CeNing Optics) were coated with SiO2 using atomic layer deposition. A film of PMMA (anisole solution, Microchem 495 PMMA A) was spincoated onto a CaF2 window (Laurell WS-400A-6NPP-LITE). Adsorbed ubiquitin and a drop-casted collagen film were used as model chiral samples. Ubiquitin is a model protein previously 12420

DOI: 10.1021/acs.jpcb.5b08176 J. Phys. Chem. B 2015, 119, 12417−12423

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Figure 5. θpp chiral SFG of the CH stretching region of an OTS SAM and drop-casted collagen. Interference and pure chiral SFG of (a) OTS and (b) collagen. Interference and chiral SFG comparison for (c) OTS and (d) collagen. Self-referencing of (e) OTS and (f) collagen. The achirality of OTS and chirality of collagen are clear in both the interference and pure chiral SFG. The self-referencing blocks the chiral signal.

Figures 5 and 6 show the θpp and θsp pure and interference chiral SFG results for the octadecyltrichlorosilane (OTS) selfassembled monolayer and drop-casted collagen. The results are qualitatively similar to the PMMA and ubiquitin results. OTS displays no chirality in interference and pure chiral SFG, while collagen has strong chirality. Figure 6 shows only the OTS results for the θsp. The collagen sample was contaminated before the θsp chiral SFG could be collected.

(Newport) was used for reproducible height and rotation during insertion of the polarizer.

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RESULTS In Figures 3−6, we demonstrate our method using the CH stretch vibrations of two model achiral samples (spin-coated PMMA film and self-assembled monolayer of octadecyltrichlorosilane) and two model chiral samples (adsorbed ubiquitin and drop-casted collagen film). In each of Figures 3−6, pure and interference chiral SFG are demonstrated in the first row, interference and pure chiral SFG are compared in the second row, and self-referencing of each sample is shown in the third row. Figures 3 and 5 show the results for the θpp polarization combinations, while Figures 4 and 6 show the results for the θsp polarization combinations. The interference and pure chiral SFG results were collected with the setups shown in Figures 1 and 2, respectively. As seen in Figure 3, the achiral PMMA film produces virtually identical +45°pp and −45°pp interference chiral SFG signals, a strong ppp pure achiral SFG signal, and no spp pure chiral SFG signal. The chiral ubiquitin produces dramatically different +45°pp and −45°pp interference chiral SFG signals, a strong ppp pure achiral SFG signal, and a clear spp pure chiral SFG signal. The corresponding results for the θsp polarizations for PMMA and ubiquitin in Figure 4 are qualitatively similar.

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DISCUSSION The middle rows of Figures 3−6 (i.e., 3c,d, 4c,d, 5c,d, and 6b) compare the pure chiral SFG signals to the interference chiral SFG signals. The teal traces are the difference between the respective +45° and −45° SFG signals, while the maroon traces are simply the pure chiral SFG polarization. For the achiral PMMA and OTS, both interference and pure methods correctly show no chirality. For the chiral ubiquitin, the pure chiral (spp and psp) signals are only positive and are similar to each other, as expected. The interference difference signals (+45° − (−45°)) produce positive and negative peaks, and the ±45°pp and ±45°sp results are not similar. The chiral collagen also shows a positive spp pure chiral signal and a ±45°pp interference difference spectrum with positive and negative phase. Like normal heterodyning, the sign of the interference chiral SFG signal reflects the phase of modes, and the 12421

DOI: 10.1021/acs.jpcb.5b08176 J. Phys. Chem. B 2015, 119, 12417−12423

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Therefore, we developed a self-referencing technique (Figure 7) that confirms the experiment’s accuracy for each sample. A

Figure 7. Detection arm for self-referencing of interference chiral SFG. The polarizer blocks the weaker chiral signal (red), transmitting the stronger achiral signal (blue). The achromatic waveplate rotates the achiral signal 45°, and the beam displacer splits the achiral signal into two equal parts, which are then simultaneously detected on the CCD camera.

polarizer after the sample eliminates the chiral polarization. The achiral signal is then rotated 45° by the waveplate and split into two equal parts by the beam displacer. After calibration for alignment and the grating efficiency (see Supporting Information), the polarizer is removed (Figure 1). Adding and removing the polarizer is the least disturbing method for referencing. With reproducible mounting positions, the polarizer has no significant effects on an achiral sample, as shown in Figures 3e, 4e, 5e, and 6c. For a chiral sample, the selfreferencing causes the chiral signal to disappear and is a quick check that any resolved differences between the +45° and −45° interference SFG signals correspond to chirality, as shown in Figures 3f, 4f, and 5f.

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CONCLUSION In conclusion, a facile method for robust self-referenced, polarization-multiplexed interference, and pure chiral SFG has been demonstrated to improve upon existing chiral SFG detection schemes. We apply polarization multiplexing to chiral SFG for the first time, facilitating simultaneous collection of orthogonal interference chiral SFG signals or of pure achiral and chiral SFG signals. For interference chiral SFG, simultaneous detection of positively and negatively interfered chiral and achiral SFG signals eliminates systematic error from alignment and laser fluctuations. For pure chiral SFG, replacing the SFG polarizer with the beam displacer allows all generated sum frequency to be collected and eliminates error from leakthrough. Self-referencing ensures that chiral signals are truly purely chiral by blocking the weak chiral signal and validating that the achiral signal splits into two equal parts. Selfreferencing, polarization-multiplexed chiral SFG increases signal-to-noise, reduces the detection time, and provides robustness to both interference and pure chiral SFG results.

Figure 6. θsp chiral SFG of OTS SAM. (a) Interference and pure chiral SFG. (b) Interference and chiral SFG comparison. (c) Selfreferencing. The achirality of OTS is clear in both the interference and pure chiral SFG.

magnitude is proportional to the chiral susceptibility. However, unlike in normal heterodyning, where the stronger reference signal is a slowly varying nonresonant signal with a constant phase, the stronger reference signal in interference chiral SFG is the achiral SFG signal containing resonant peaks with a variable phase across the spectrum. Therefore, while interference chiral SFG has better signal-to-noise ratios than pure chiral SFG making it easier to detect chiralityand gives more information than pure chiral SFG, the complicated phase information must be analyzed carefully because it is in relation to the achiral signal. Increasing the signal-to-noise ratio of interference chiral SFG depends on establishing the noise level for the difference between the +45° and −45° interference signals. Similarly, the pure chiral experiment must make certain that the stronger achiral signal is not leaking into the chiral polarization detection.20 In testing, we found that the chiral SFG signals were very sensitive to sample position, pointing, and other technical details. As such, minute alignment changes caused by switching between the sample and gold reference were enough to cause significant erroneous differences between the +45° and −45° interference SFG signals even for achiral samples.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b08176. Specifics details about the self-referencing setup for pure chiral SFG spectroscopy and correcting for alignment imperfections (PDF) 12422

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(14) Fu, L.; Wang, Z.; Yan, E. C. Y. Chiral Vibrational Structures of Proteins at Interfaces Probed by Sum Frequency Generation Spectroscopy. Int. J. Mol. Sci. 2011, 12, 9404−9425. (15) Tong, Y.; Zhao, Y.; Li, N.; Osawa, M.; Davies, P. B.; Ye, S. Interference Effects in the Sum Frequency Generation Spectra of Thin Organic Films. I. Theoretical Modeling and Simulation. J. Chem. Phys. 2010, 133, 034704. (16) Wang, H.-F.; Velarde, L.; Gan, W.; Fu, L. Quantitative SumFrequency Generation Vibrational Spectroscopy of Molecular Surfaces and Interfaces: Lineshape, Polarization, and Orientation. Annu. Rev. Phys. Chem. 2015, 66, 189−216. (17) Anglin, T. C.; Massari, A. M. Polarization-Multiplexed Vibrational Sum Frequency Generation for Comprehensive Simultaneous Characterization of Interfaces. Opt. Lett. 2012, 37, 1754−1756. (18) Belkin, M. A.; Kulakov, T. A.; Ernst, K.-H.; Yan, L.; Shen, Y. R. Sum-Frequency Vibrational Spectroscopy on Chiral Liquids: A Novel Technique to Probe Molecular Chirality. Phys. Rev. Lett. 2000, 85, 4474−4477. (19) Stokes, G. Y.; Gibbs-Davis, J. M.; Boman, F. C.; Stepp, B. R.; Condie, A. G.; Nguyen, S. T.; Geiger, F. M. Making “Sense” of DNA. J. Am. Chem. Soc. 2007, 129, 7492−7493. (20) Fu, L.; Zhang, Y.; Wei, Z.; Wang, H. Intrinsic Chirality and Prochirality at Air/R-(+)- and S-(−)-Limonene Interfaces: Spectral Signatures With Interference Chiral Sum-Frequency Generation Vibrational Spectroscopy. Chirality 2014, 26, 509−520. (21) Pászti, Z.; Wang, J.; Clarke, M. L.; Chen, Z. Sum Frequency Generation Vibrational Spectroscopy Studies of Protein Adsorption on Oxide-Covered Ti Surfaces. J. Phys. Chem. B 2004, 108, 7779−7787. (22) Han, Y.; Hsu, J.; Ge, N.-H.; Potma, E. O. Polarization-Sensitive Sum-Frequency Generation Microscopy of Collagen Fibers. J. Phys. Chem. B 2015, 119, 3356−3365. (23) Rocha-Mendoza, I.; Yankelevich, D. R.; Wang, M.; Reiser, K. M.; Frank, C. W.; Knoesen, A. Sum Frequency Vibrational Spectroscopy: The Molecular Origins of the Optical Second-Order Nonlinearity of Collagen. Biophys. J. 2007, 93, 4433−4444.

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (P.B.P.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project is funded by the Arnold and Mable Beckman Foundation through a Young Investigator Award. Aliyah Barrett prepared the OTS self-assembled monolayers and PMMA with use of the Cornell Center for Materials Research Shared Facilities, which are supported through the NSF MRSEC program (DMR-1120296), and the Cornell NanoScale Facility, a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (ECCS-0335765).

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ABBREVIATIONS SFG, sum frequency generation; OTS, octadecyltrichlorosilane; PMMA, poly(methyl methacrylate); CCD, charge-coupled device.

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REFERENCES

(1) Belkin, M. A.; Shen, Y. R. Non-Linear Optical Spectroscopy as a Novel Probe for Molecular Chirality. Int. Rev. Phys. Chem. 2005, 24, 257−299. (2) Eisenthal, K. B. Liquid Interfaces Probed by Second-Harmonic and Sum-Frequency Spectroscopy. Chem. Rev. 1996, 96, 1343−1360. (3) Wang, J.; Chen, X.; Clarke, M. L.; Chen, Z. Detection of Chiral Sum Frequency Generation Vibrational Spectra of Proteins and Peptides at Interfaces in Situ. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 4978−4983. (4) Fu, L.; Ma, G.; Yan, E. C. Y. In Situ Misfolding of Human Islet Amyloid Polypeptide at Interfaces Probed by Vibrational Sum Frequency Generation. J. Am. Chem. Soc. 2010, 132, 5405−5412. (5) Fu, L.; Liu, J.; Yan, E. C. Y. Chiral Sum Frequency Generation Spectroscopy for Characterizing Protein Secondary Structures at Interfaces. J. Am. Chem. Soc. 2011, 133, 8094−8097. (6) Okuno, M.; Ishibashi, T. Chirality Discriminated by HeterodyneDetected Vibrational Sum Frequency Generation. J. Phys. Chem. Lett. 2014, 5, 2874−2878. (7) Nguyen, K. T. An Electronically Enhanced Chiral Sum Frequency Generation Vibrational Spectroscopy Study of Lipid-Bound Cytochrome c. Chem. Commun. 2015, 51, 195−197. (8) Meister, K.; Lotze, S.; Olijve, L. L. C.; DeVries, A. L.; Duman, J. G.; Voets, I. K.; Bakker, H. J. Investigation of the Ice-Binding Site of an Insect Antifreeze Protein Using Sum-Frequency Generation Spectroscopy. J. Phys. Chem. Lett. 2015, 6, 1162−1167. (9) Haupert, L. M.; Simpson, G. J. Chirality in Nonlinear Optics. Annu. Rev. Phys. Chem. 2009, 60, 345−365. (10) Wang, H.-F. In Situ Measurement of Chirality of Molecules and Molecular Assemblies with Surface Nonlinear Spectroscopy. In Comprehensive Chiroptical Spectroscopy: Instrumentation, Methodologies, and Theoretical Simulations; John Wiley & Sons, Inc.: New York, 2011; Vol. 1, pp 373−407. (11) Wei, F.; Xu, Y.; Guo, Y.; Liu, S.; Wang, H. Quantitative Surface Chirality Detection with Sum Frequency Generation Vibrational Spectroscopy: Twin Polarization Angle Approach. Chin. J. Chem. Phys. 2009, 22, 592−600. (12) Simpson, G. J. Molecular Origins of the Remarkable Chiral Sensitivity of Second-Order Nonlinear Optics. ChemPhysChem 2004, 5, 1301−1310. (13) Yan, E. C. Y.; Fu, L.; Wang, Z.; Liu, W. Biological Macromolecules at Interfaces Probed by Chiral Vibrational Sum Frequency Generation Spectroscopy. Chem. Rev. 2014, 114, 8471− 8498. 12423

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