Biomedical Engineering Reference
In-Depth Information
oriented and, therefore, they do not produce coherent SHG, resulting in a high-contrast SHG image, in
which only the cell membranes are visible.
This mechanism of high-contrast imaging has been extensively used, in combination with voltage-
sensitive dyes, to achieve improved sensitivity in the optical measurement of membrane potential
(Stuart and Palmer, 2006; Wilt et al., 2009). In fact, a strong limitation of conventional membrane imag-
ing techniques derives from the reduction in the measured dye response to membrane potential varia-
tions due to molecules that are in the focal volume but are not embedded in the plasma membrane, thus
producing background. Variations in this background have the effect of making the observed signal
difficult to quantify in terms of membrane potential. Because of the molecular alignment required for
SHG, signal only emanates from properly oriented dye molecules in the membrane; thus, its effective
membrane potential response is not significantly attenuated by background, allowing for a more quan-
titative relation between optical signal changes and membrane potential variations.
Beyond this fundamental background reduction, coherent summation also provides an order-based
mechanism for membrane potential sensing. In fact, two mechanisms contribute to the voltage sensitiv-
ity: an electro-optic-induced alteration of the molecular hyperpolarizability and an electric-field-induced
alteration of the degree of molecular alignment (Moreaux et al., 2003; Pons et al., 2003). The latter was
experimentally characterized by SPA measurements showing that, with increasing electric field across
the membrane, the embedded dipolar molecules reduce their angular dispersion, increasing the coherent
summation.
The extreme sensitivity of SHG to dipole alignment in the membrane was demonstrated also by the
disappearance of SHG signal upon fertilization in sea urchin eggs stained with di-8-ANEPPS (Millard
et al., 2005): the production of micro-villi in the membrane, in fact, breaks the regular arrangement of
the dye molecules in the plane of the membrane itself, leading to a dramatic reduction of SHG.
5.5 endogenous SHG imaging
As described above, the condition for a nonzero value of first hyperpolarizability in a molecule is the
presence of an asymmetry of charge distribution spanned by a uni-axial charge transfer path.
Many biological molecules have this property, leading to the interesting prospective that biological
samples may be rich of endogenous HRS emitters. In fact, the possibility of observing HRS in biomol-
ecules was demonstrated more than 40 years ago, when SHG from amino acid crystals was detected
(Rieckhoff, 1965). The ubiquitous distribution of amino acids in biological samples, therefore, insures
the presence of HRS emitters in every cell and tissue. However, as illustrated above, a proper spatial
organization of the HRS emitters is required for SHG, so that endogenous signal could arise, for exam-
ple, in biological samples with semi-crystalline organization.
The first SHG biological imaging was reported by Freund et al. on connective tissue (Freund and
Deutsch, 1986). In this tissue, in fact, the structural organization of collagen in fibrils and fibers disposes
the HRS emitters in a lattice leading to coherent summation, that is, to SHG. Figure 5.7 shows SHG
images of rat-tail tendon collagen taken at the dawn of SHG microscopy (panel a) and today (panel b).
Owing to the peculiarity of SHG (i.e., label-free micron-scale resolution in deep tissue), this imaging
technique has tremendous potential for biomedical applications, ranging from morphological charac-
terization of healthy and pathological connective tissue
in vivo
(Brown et al., 2003; Cicchi et al., 2007;
Cicchi et al., 2010; Han et al., 2008; Wang et al., 2007) to quantitative measurement of fibril orientation
within the pixel size (Stoller et al., 2002; Williams et al., 2005). Collagen type I is the most abundant
protein in mammals, featuring a hierarchical structure ranging from the atomic and molecular scale
to the macroscopic scale. It consists of tropo-collagen molecules of an approximate length of 280 nm
and a diameter of 1.5 nm, staggered side-by-side to form fibrils. Fibrils can be considered the unitary
structures constituting collagen. They are approximately 1 μm in length and 30 nm in diameter and
they hierarchically organize themselves in bigger fibers or sheets in order to form collagen fiber bundles
in skin dermis or lamellae in cornea.
