Second-harmonic phase determination by real-time in situ interferometry.

Second Harmonic Generation (SHG) has emerged as a highly sensitive probe of protein conformation. SHG can also be used to determine the tilt angle of an SHG-active moiety bound to a surface-adsorbed protein through polarization-dependent measurements. However, due to the coherent nature of SHG, interference occurs between the SHG produced by the SHG-active moieties and background sources at a solid-liquid interface, obscuring the signal of interest. In order to separate the protein-specific signal from the background signal, the phase difference between these two different sources of SHG must be determined. Although the phase difference can be obtained through a conventional interferometric approach involving a phase-modulated SHG source external to the sample, it can be sensitive to drift and other instabilities. We present here a simple, convenient, and crucially, model-independent method to determine the phase difference for any system in which the intensity of SHG-active moieties can be varied. We demonstrate the approach with time-resolved measurements of an SHG-active labeled protein binding to a supported lipid bilayer surface using a total internal reflection (TIR) geometry. This approach requires no additional optics beyond what is required to measure SHG and is highly stable since the interferometry occurs in situ, within the sample over a nanometer length scale, rather than external to it. To validate our measurements and the general approach, we constructed a dual-beam, external SHG interferometer in a TIR geometry. We also validated our approach by applying the in situ method to previously published measurements of the phase difference, obtaining the same values without recourse to a specific adsorption model.