Second harmonic generation is an excellent tool for probing interfaces and surfaces. Second harmonic generation is a nonlinear optical approach in which two photons with a fundamental frequency are converted to a single photon of twice the fundamental frequency. The probability of successful conversion is generally small, requiring single photon counting detectors. Owing to the small signals involved, background sources affect the magnitude of the signal. Since the background second harmonic generation is coherent with the signal of interest, it becomes important to acquire phase details of the two sources in order to parse them.
Interference between the background second harmonic generation sources and signals of interest have been adopted for silicon and silicon oxide surfaces, dye-labelled proteins immobilized on glass, and dyes adsorbed onto glass. Various strategies have been adopted to eliminate the effect of the background second harmonic generation from the total signal through phase difference measurements between the two. These approaches, however, present some drawbacks.
An interferometer is challenging to implement in total internal reflection optical systems because of dispersion in the coupling prism. A Langmuir model yields inaccurate results when the underlying kinematics are different from the ideal behavior. Therefore, Bason Clancy and Joshua Salafsky at Biodesy Inc. in California developed a simple and model-independent approach to measure the phase difference of any system in which the magnitude of the second harmonic signal from second-harmonic active moieties may be varied. The approach is demonstrated by implementing time-resolved measurements of second harmonic generation-active labeled protein attached to a lipid surface using the geometry of total internal reflection. Their work is now published in Physical chemistry, Chemical physics.
In the first method, the authors used signals from the second harmonic generation active dye-labelled protein and background sources at the solid-liquid interface to build anin situ interferometer. They controlled the amplitude of one signal with respect to the other in order to determine the phase difference.
In a bid to authenticate the outcomes of the proposed in situ approach, the researchers built a second harmonic generation interferometer which could be employed with dispersive elements, for example, a prism in a total internal reflection orientation. This design implements dual fundamental beams to generate signal and auxiliary second harmonic beams simultaneously on a selected sample. By increasing the path length of a beam with respect to the other, the authors were able to modulate the phase between the two beams in the developed interferometer. A rotatable coverslip was necessary to achieve this. When the coverslip was rotated, the path length of the beam through the glass increased, therefore changing the phase relationship between the two beams.
The in situ method was faster, model-independent and produced accurate results. Both beams interfered within the sample instead of external to it, making the method insensitive to external fluctuations. Interference curves were generated faster allowing high throughput phase determination. This was an advantage for different dye sites on particular protein, where local effects could change nonlinear susceptibility, and consequently the phase.
After phase measurement, precise background subtraction was possible. The effect of the dye-labelled signal could be separated from the total signal. This allowed for precise calculation of the protein-only intensity, paving the way for sensitive measurements of the angular arrangement of the dye label and, in turn, of protein structure via polarization-dependent experiments.
Bason Clancy and Joshua Salafsky. Second-harmonic phase determination by real-time in situ interferometry. Physical Chemistry Chemical Physics 2017, 19, pages 3722—3728.
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