Conclusions

High resolution optical waveguide lightmode spectroscopy (HR-OWLS), also known as high resolution molecular microscopy (HRMM) is able to yield detailed physico-chemical characterization of proteins, other biopolymers, polymers and other particles (even living cells (Ramsden et al. 1995)) adsorbing at and desorbing from buried surfaces such as the solid/liquid interface.

The high precision of OWLS—in essence due to its very high signal/noise ratio, traceable to the very large number of multiple reflections per unit area ofobserved interface—enables the opto-geometric parameters ofeven monomolecular layers to be determined, such as the thickness (to a precision of ±10-1nm) and the refractive indices (to a precision of about ±3x 10-4), from which a robust determination of the predominant orientation of the molecular components of the layer can be deduced. Small conformational changes resulting from a change of conditions can be detected in this way.

Much more information is obtainable if the kinetics of addition and spontaneous removal of particles to and from the surface is followed with good time resolution: the mean projected area and shape of the adsorbing particles, their lateral diffusion and sticking coefficients if they tend to cluster on the surface, the lattice parameters of two dimensional crystals if they form regular arrays, their expansion coefficient and the kinetics of expansion if they undergo area-changing reorientation or conformational change, the adsorption free energy barrier, etc. If a series of experiments under different conditions is carried out, the different contributions to the adsorption free energy barrier maybe resolvable. If the memory function can be characterized, a wealth of detail concerning the dynamics of the adsorbed protein maybe inferred, including cooperative effects. Additional experiments involving repeated pulses of protein solutions of different concentrations may further illuminate these processes.

The HRMM approach is fundamentally different from that of scanning probe or single molecule fluorescence microscopies, its main rivals in examining the behaviour of complex nanoparticles such as proteins, in that individual objects or their locations are not imaged. Hence the radial distribution function of the deposited objects cannot be measured directly. Nevertheless, all the information obtainable from the imaging techniques can be inferred from OWLS measurements, as well as a great many essential parameters characterizing all aspects of the adsorption process in a totally non-destructive, non-invasive fashion.

Acknowledgements. The author sincerely thanks the other members of the MEMbrane-coated Optical-grating Coupler Sensors (MEMOCS) consortium for numerous valuable discussions on the subject of this Chapter during the past few years.

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