Biomedical Engineering Reference
In-Depth Information
nonresonant background [28]. This requirement is most readily met by abundant C-H stretches. One
such example showing this point comes from the imaging of collagen fibrils using CARS and SFG,
where CARS had much inferior S/N than SFG (SFG is closely related to SHG and their S/N should
be comparable) [60]. When used for imaging proteins, CARS and SRS methods directly report CH
2
content of the proteins. Wang et al. indicated that when CARS imaged both elastin and collagen fibers,
simultaneously, the former showed higher contrast due to greater abundance of CH
2
groups. On the
other hand, SFG imaging only recorded collagen fibers, where elastin was completely absent [60]. The
more complex and expensive CARS/SRS microscopes have the advantage of a wider range of applica-
tions, but lack the capability of structural sensitivity of SHG/SFG. SHG/SFG imaging methods will be
more valuable for disease diagnosis, where only change of protein conformations occurs without the
variation of chemical composition. However, the modalities have great promise when used in conjunc-
tion as they provide complementary information. An overall summary of the attributes of all the NLO
modalities is given in Table 4.1.
4.5 Summary
SHG and other nonlinear optical microscopies are all imaging methods whose development resulted
from introducing nonlinear optics into laser scanning microscopy, of which multiphoton excited flu-
orescence imaging is the most straightforward conceptually. While all the NLO imaging methods
have many similarities experimentally and their nonlinear nature enable their 3D capability to image
biological samples, the unique aspects of SHG microscopy arise because of its sensitivity to protein
microscale structures, especially to those of collagen fibrils/fibers. Given the numerous and diverse
pathologies that have alterations in the collagen assembly, SHG has a high potential to develop into a
clinical diagnostic imaging tool. However, the physics behind its structural sensitivity also limits its
application generality.
The NLO imaging techniques are often incorporated into one microscope as complementary modali-
ties to combine the merits of the different contrast mechanisms. This field is still undergoing rapid devel-
opment, with the current technologies finding broader applications, with new techniques to improve the
current technologies (such as NLO endoscopies, and manipulating laser polarizations for the benefit of
information extraction or noise suppression), and sometimes with completely new technologies starting
to emerge (e.g., stimulated emission microscopy). NLO microscopies have already significantly pushed
forward scientists' ability to understand academic questions about cells and organisms, and will likely
revolutionize clinical imaging approaches for disease diagnosis.
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