An EKSPLA Nd:YAG laser, operating at 50 Hz, was used to generate both a fixed visible (532 nm) and tunable IR beam (1000–4000 cm−1) via sequential pumping through an EKSPLA optical parametric generation/amplification and difference frequency unit. The optical parametric generation/amplification unit utilized a barium borate crystal, while the difference frequency unit used an AgGaS2 crystal. The visible and IR beams (∼150 μJ/pulse) were overlapped spatially and temporally at the aqueous-air interface to generate SFG photons, which were spectrally filtered, dispersed by a monochromator, and detected with a gated photomultiplier tube. Both beams were focused to a ∼1 mm diameter at the interface. Spectra were collected in 4 cm−1 steps with 100 acquisitions per step. Three consecutive spectra were collected and averaged for the lipid monolayer before and after the NPs interacted with the lipid monolayer. SFG spectra were collected in polarization combination—ssp (s-polarized SFG, s-polarized visible, p-polarized IR) in the C–D vibrational region (2000–2300 cm−1). SFG spectra were normalized by dividing the collected signal at each wavelength by the visible and infrared beam intensities. SFG spectra were fit with the following Lorentzian line-shape expansion: I(ω)∝|χNR(2)eiφ+∑nAnω−ωn+iΓn2|2,(1)where I is the intensity Γn, An, and ωn are the full width at half max, amplitude, and resonant frequency of the nth vibrational mode, respectively, χNR(2) is the nonresonant background and φ is the phase of the nonresonant background.30,4630. J. D. Pickering, M. Bregnhøj, A. S. Chatterley, M. H. Rasmussen, K. Strunge, and T. Weidner, Biointerphases 17, 011201 (2022). https://doi.org/10.1116/6.000140146. A. P. Carpenter and J. E. Baio, Biointerphases 17, 031201 (2022). https://doi.org/10.1116/6.0001844 The order ratio is calculated by taking the ratio between the amplitudes of the methyl symmetric stretch (CD3 ss) and the methylene symmetric stretch (CD2 ss).47,4847. L. Qiao, A. Ge, M. Osawa, and S. Ye, Phys. Chem. Chem. Phys. 15, 17775 (2013). https://doi.org/10.1039/c3cp52484a48. A. G. Lambert, P. B. Davies, and D. J. Neivandt, Appl. Spectrosc. Rev. 40, 103 (2005). https://doi.org/10.1081/ASR-200038326 It is important to note that in SFG, depending on the molecular bond, An can have a positive or negative value. In the homodyne SFG intensity spectrum, where the intensity is proportional to the square of the sum frequency electric field, the absolute orientational information is lost in the squaring of the electric field.4949. R. E. Pool, J. Versluis, E. H. G. Backus, and M. Bonn, J. Phys. Chem. B 115, 15362 (2011). https://doi.org/10.1021/jp2079023 However, when fitting SFG spectra, the sign of An becomes relevant and is related to the interference of the resonances. In general, the symmetric and asymmetric vibrations have orthogonal dipoles, thus they are assigned an amplitude opposite to one another.4949. R. E. Pool, J. Versluis, E. H. G. Backus, and M. Bonn, J. Phys. Chem. B 115, 15362 (2011). https://doi.org/10.1021/jp2079023 The data presented in Fig. 3 and in Table II are representative spectra and fitting parameters for the spectra, respectively. Replicate experiments are performed for each type of experiment, and an average and standard deviation are calculated.