Biomedical Engineering Reference
In-Depth Information
Kwan et al., 2008). We describe a method for interpreting SHG anisotropy in terms of molecular confor-
mation 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.
The description of molecular structural dynamics occurring in a living cell and determining its biology
requires a variety of techniques to encompass the spatial and temporal scales involved. In fact, while x-ray
crystallography provides static structures with atomic resolution, complementary techniques are necessary
to probe structural dynamics in the cell. A good example of use of complementary approaches to the study
of structure−function relationship is represented by skeletal muscle. In fact, due to its structural organiza-
tion, muscle tissue has long been a sample of choice for the development and application of novel biophysi-
cal methodologies. Knowledge of the atomic structures of myosin and actin, combined with information
from cryo-EM (Piazzesi et al., 2007; Reedy, 2000), electron paramagnetic resonance (Thomas et al., 2009),
x-ray diffraction (Huxley, 2004), fluorescence polarization (Corrie et al., 1999), birefringence (Irving, 1993;
Peckham et al., 1994) has led to a structural description of the chemo-mechanical energy conversion in
terms of the lever-arm hypothesis of the working stroke in the myosin motor (Holmes, 1997; Rayment et al.,
1993). In this chapter, we illustrate the capability of SHG to probe myosin molecular conformation
in vivo
.
SHG is a nonlinear second-order optical process occurring in systems without a center of symmetry
and which have a large molecular hyperpolarizability. This condition is easily fulfilled at the molecular
level by a moiety in which an electron donor is connected to an electron acceptor by a π-conjugated sys-
tem. Further, only systems with non-centrosymmetric packing and organization of the dipoles present
large second-order susceptibility and can generate SHG. Examples of such symmetries are biological
membranes, where only one leaflet is stained with a donor−acceptor molecule.
In proteins, the intrinsic SHG sources lie within the amide bonds of polypeptide chains and the
peptide bond C−N between two amino acids can be considered as the elementary dipole (Conboy and
Kriech, 2003; Mitchell et al., 2005). Hence, the capability of a protein to generate SHG depends on its
three-dimensional structure and folding which determine the degree of alignment of peptide bonds.
In secondary structures like helices the peptide bonds are very well ordered and, therefore, their har-
monophoric units generate coherent second-harmonic signal leading to a constructive interference. The
tertiary structure of the protein can also influence the SHG efficiency because the helices inside the
protein may be aligned or oriented in random directions. SHG signal is experimentally observed only
from proteins which have ordered structure: myosin which is mainly constituted by α-helices; collagen
which is constituted by a triple-helix and microtubules.
The characterization of the SHG physical basis will be described in the following sections and will
demonstrate the exquisite sensitivity of SHG to order and molecular organization. Further, employing a
full reconstruction of the contributing elementary SHG emitters at atomic scale, we provide a molecular
interpretation of the SHG measurements in terms of structural conformation and degree of organiza-
tion of the proteins
in vivo
.
In Section 5.2, we describe the physical principle underlying the production of SHG through coher-
ent summation. This description represents the theoretical basis for all subsequent applications of SHG
microscopy to the study of structural order and molecular conformation
in vivo
. Most applications
rely on the polarization properties of SHG. Section 5.3 provides a full mathematical framework for the
analysis of SHG polarization anisotropy data in terms of the orientation distribution of HRS emitters
within the focal volume. Section 5.4 shows how the dependence of sensitivity of SHG on molecular order
is exploited in a simple geometry provided by the membrane lipid bilayer leading to high contrast mem-
brane imaging. The sensitivity of SHG to molecular order also leads to intrinsic signal from specific tis-
sues characterized by ordered lattices of proteins; the most prominent examples are described in Section
5.5; the source of HRS within proteins is then described in Section 5.6. With the concepts illustrated in
Sections 5.2 and 5.3, and the molecular origin of HRS described in Section 5.6, Section 5.7 provides an
explanation of the methods through which SHG polarization anisotropy can be used to probe molecular
order and conformation within living tissues.
