Biomedical Engineering Reference
In-Depth Information
5
Molecular Structure and
Order with Second-
Harmonic Generation
Microscopy
Francesco Vanzi
European Laboratory for
Non-Linear Spectroscopy
(LENS)
University of Florence
Leonardo Sacconi
European Laboratory for
Non-Linear Spectroscopy
(LENS)
National Institute of
Optics—National Research
Council (INO— CNR)
Riccardo Cicchi
National Institute of
Optics—National Research
Council (INO— CNR)
5.1 Introduction ......................................................................................103
5.2 From Hyper Rayleigh Scattering to SHG......................................105
5.3 SHG Polarization Anisotropy.........................................................106
5.4 Membrane SHG Imaging ................................................................109
5.5 Endogenous SHG Imaging .............................................................. 111
5.6 Source of the Endogenous SHG...................................................... 116
5.7 Probing Protein Structural Conformation ................................... 118
5.8 Conclusions........................................................................................120
References......................................................................................................120
Chiara Stringari
University of California,
Irvine
Francesco S. Pavone
European Laboratory for
Non-Linear Spectroscopy
(LENS)
University of Florence
5.1 introduction
Advances in imaging technologies drive a constant progress in our capability of probing structures and
their dynamics within cells and tissues. The application of nonlinear spectroscopy to optical micros-
copy (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. The coherent nature of second-harmonic
generated light (Campagnola and Loew, 2003; Moreaux et al., 2001) makes this optical process intrinsi-
cally 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 prin-
ciples 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 emit-
ters 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;
103
