Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans Original Research Article

Second-harmonic generation (SHG) is applied to problems of probing membrane proteins and functionally imaging around selective sites and at single molecules in biological membranes. The membrane protein bacteriorhodopsin (bR) has been shown to have large second-harmonic (SH) intensities that are modulated by protein/retinylidene chromophore interactions. The nonlinear optical properties of model compounds, which simulate these protein chromophore interactions in retinal proteins, are studied in this work by surface SHG and by hyper-Rayleigh scattering. Our results indicate that non-conjugated charges and hydrogen bonding effects have a large effect on the molecular hyperpolarizability of the retinal chromophore. However, mbR, the model system studies suggest that polarizable amino acids strongly affect the vertically excited state of the retinylidene chromophore and appear to play the major role in the observed protein enhancement (>50%) of the retinylidene chromophore molecular hyperpolarizability and associated induced dipole. Furthermore, the data provide insights on emulating these interactions for the design of organic nonlinear optical materials. Our studies have also led to the development of dyes with large SH intensities that can be embedded in cell membranes and can functionally image membrane potential. Single molecules of such dyes in selected single molecular regions of a cell membrane have been detected. SHG from green fluorescent protein (GFP) selectively expressed in concert with a specific protein in neuronal cells in a transgenic form of the worm C. elegans is also reported. The membrane potential around the GFP molecules expressed in these cells has been imaged with SHG in live animals.