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. e application of nonlinear spectroscopy to optical microscopy (Denk et al., 1990; Helmchen and Denk, 2005; Zipfel et al., 2003b) has led to new perspectives both in basic research and in the potential development of very powerful noninvasive diagnostic tools. Some of these techniques permit optical probing of biological functions (Dombeck et al., 2004; Skala et al., 2007; Svoboda and Yasuda, 2006; Zipfel et al., 2003a), as well as monitoring molecular structure and dynamics in vivo (Nucciotti et al., 2010). 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. e coherent nature of second-harmonic generated light (Campagnola and Loew, 2003; Moreaux et al., 2001) 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 (Moreaux et al., 2000; Pons et al., 2003; Sacconi et al., 2005). Applications of these principles range from voltage-sensitive membrane imaging via exogenous labeling (Dombeck et al., 2005; Jiang et al., 2007; Millard et al., 2003; Moreaux et al., 2003; Nuriya et al., 2006; Sacconi et al., 2006a, 2008) to probing order and structural organization in tissues rich in intrinsic second-harmonic emitters such as collagen (Brown et al., 2003; Cox et al., 2003; Freund and Deutsch, 1986; Jain et al., 2003; Stoller et al., 2002; Williams et al., 2005), myosin (Both et al., 2004; Campagnola et al., 2002; Plotnikov et al., 2008; Plotnikov, 2006; Vanzi et al., 2006), tubulin (Campagnola et al., 2002; Dombeck et al., 2003; Kwan et al., 2008). 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 noninvasively probing molecular structure and order in vivo.