Molecular Structure and Order with Second-Harmonic Generation Microscopy

Advances in imaging technologies drive a constant progress in our capability of probing structures and their dynamics within cells and tissues. The application of non-linear spectroscopy to optical microscopy has led to new perspectives both in basic research and in the potential development of very powerful non-invasive diagnostic tools. Some of these techniques permit optical probing of biological functions, as well as monitoring molecular structure and dynamics in vivo. In this chapter we review the properties of second-harmonic generation (SHG) and its application for the characterization of biological samples in terms of degree of molecular order, structural organization and dynamics. The coherent nature of second-harmonic generated light makes this optical process intrinsically sensitive to the angular distribution of the emitting elements in the focal volume, allowing both high-contrast imaging of ordered versus disordered structures and quantitative analysis of molecular orientation. Applications of these principles range from voltage-sensitive membrane imaging via exogenous labeling to probing order and structural organization in tissues rich of intrinsic second-harmonic emitters such as collagen, myosin, tubulin. We describe a method for interpreting SHG anisotropy in terms of molecular conformation of the emitting proteins within a living tissue. Due to the properties of nonlinear spectroscopy, this method empowers SHG microscopy with the unique advantage of non-invasively probing molecular structure and order in vivo.