Biomedical Engineering Reference
In-Depth Information
TABLE 18.1
SHG-Active Materials of Interest in
Tissue Engineering Listed with Their Function
Material
Function in Tissue Engineering
Collagen
Scaffold material, ECM component
Cellulose
Scaffold material
Silk
Scaffold material
Myosin
Muscle tissue component
These limitations of established microscopy techniques make nondestructive, label-free methods for
three-dimensional monitoring of unmodified cell-scaffold constructs of high interest. SHG microscopy,
introduced for the first time in the 1980s (Freund and Deutsch 1986), has in the last decade become
a widely used imaging method benefitting from developments in laser and microscope technology
(Campagnola et al. 2002, Campagnola and Loew 2003, Zipfel et al. 2003). SHG microscopy permits spe-
cific label-free visualization with high spatial resolution in three dimensions probing nonlinear optical
properties in structures having noncentrosymmetric molecular arrangement. Although being limited
to monitoring molecules of specific symmetry properties (and their assembly), this technique is a valu-
able characterization method in tissue engineering since a number of materials of interest are SHG-
active. Table 18.1 presents these materials and their functions in tissue engineering.
A number of publications have emerged in later years using SHG as well as other nonlinear microscopy
methods for tissue characterization and review papers addressing applications to tissue engineering have
also been presented (Georgakoudi et al. 2008, Schenke-Layland 2008). In addition, technical aspects of
importance for SHG microscopy in tissue have been investigated, such as polarization effects (Matcher
2009) and phase-matching considerations (LaComb et al. 2008). The usage of microscopy techniques
based on nonlinear optics is promoted by the development of smaller, more user-friendly, low-cost, short-
pulse laser systems (Svedberg et al. 2010, Tang et al. 2009). In particular, combining SHG microscopy
with other techniques, such as multi-photon fluorescence, coherent anti-Stokes Raman scattering (CARS)
(Cheng 2007), and THG (Yu et al. 2007), valuable insights in the relationship between architecture and
function of artificial tissue constructs can be obtained. Thus, SHG microscopy is a valuable instrument
within tissue engineering and an increased future use of the technique can be expected. In the following
sections, some examples showing the potential of the technique will be presented.
18.2 SHG Microscopy on collagen in tissue engineering
Collagen is a class of natural proteins containing more than 20 identified types with the fibrillar type
I being the most abundant and the major component of connective tissue. The primary unit of col-
lagen type I, tropocollagen, consists of three polypeptides arranged in a right-handed triple helix for-
mation, typically 300 nm long and 1 nm wide, stabilized by hydrogen bonds. Tropocollagen units are
able to self-organize into arrays and can then form larger structures such as microfibrils, fibrils, and
fascicles. The ordering of the material structure makes it highly SHG-active, resulting in strong signals
(Campagnola et al. 2002), and the SHG process in collagen type I fibers has been well characterized
(Stoller et al. 2002, Williams et al. 2005). SHG microscopy has been compared to established techniques
for collagen imaging such as histochemical staining with Masson's Trichrome dye and fluorescence
microscopy using Sirius Red dye (Cox et al. 2003). Detailed comparison between corresponding images
showed that it was possible to identify finer collagen fibrils by SHG microscopy, not visualized using
the conventional methods. Furthermore, Sirius Red to some degree also labeled other, less-crystalline
types of collagen and thus showed less specificity to collagen type I than SHG. The van Giesen stain is an
alternative method to label and visualize collagen. A comparison between this stain and SHG micros-
copy of fibrous liver tissue showed good overall agreement with the advantage for SHG in imaging small
collagen structures (Brackmann et al. 2010b).
