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

Bason Clancy1, Joshua Salafsky1

  • 1Biodesy Inc., 384 Oyster Point Blvd., Suite 8, South San Francisco, CA 94080, USA. joshua.salafsky@biodesy.com.

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Summary

Researchers developed a stable, model-independent method to measure the phase difference in Second Harmonic Generation (SHG) signals from surface-adsorbed proteins. This technique enhances the analysis of protein conformation and tilt angles at solid-liquid interfaces.

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Area of Science:

  • Biophysics
  • Surface Science
  • Spectroscopy

Background:

  • Second Harmonic Generation (SHG) is a sensitive technique for probing protein conformation and molecular orientation at interfaces.
  • Interference between SHG signals from surface-adsorbed proteins and background sources complicates analysis.
  • Determining the phase difference between these SHG sources is crucial for isolating the protein-specific signal.

Purpose of the Study:

  • To develop a simple, convenient, and model-independent method for determining the phase difference in SHG signals.
  • To overcome limitations of conventional interferometric approaches, such as sensitivity to drift and instability.
  • To enable accurate measurement of protein conformation and tilt angles at solid-liquid interfaces.

Main Methods:

  • A novel in situ interferometric method was developed to measure the phase difference by varying the intensity of SHG-active moieties.
  • The approach was demonstrated using time-resolved measurements of an SHG-active labeled protein binding to a supported lipid bilayer.
  • Total internal reflection (TIR) geometry was employed, requiring no additional optics beyond standard SHG instrumentation.

Main Results:

  • The in situ method successfully determined the phase difference without relying on specific adsorption models.
  • The approach demonstrated high stability due to in situ interferometry occurring at the nanometer scale within the sample.
  • Validation was achieved by comparing results with a conventional external SHG interferometer and previously published data.

Conclusions:

  • The presented model-independent, in situ method provides a robust and stable approach for phase difference determination in SHG.
  • This technique significantly improves the ability to analyze protein conformation and orientation at solid-liquid interfaces.
  • The method offers a practical advancement for biophysical studies utilizing SHG spectroscopy.