Biomedical Engineering Reference
In-Depth Information
In this chapter, we review (i) the fundamental properties of the different nonlinear optical tech-
nologies used to study the arterial wall; (ii) their application to identify the different macromolecular
microstructures that comprise the arterial vascular bed; and (iii) the insights gained from these studies
regarding the role that these microstructures play in arterial health and disease. In addition to second
harmonic generation (SHG), we will review these other multiphoton excitation schemes since the infor-
mation they provide is critical for the interpretation of vascular wall images.
13.2 Methodology and Preparations
13.2.1 nonlinear optical Applications in the Vessel Wall
As mentioned in this chapter's Introduction, optical sectioning in multiphoton microscopy [1,2] allows
improved collection efficiency over single-photon confocal microscopy for several reasons. First, the pri-
mary excitation light at a lower energy (generally IR) is less susceptible to scattering and absorbance, which
allows deeper tissue penetration. Second, the multiphoton effect intrinsically restricts emission to the focal
spot and maintains optical sectioning without confocal detection. Thus, every emitted photon that escapes
from the tissue can be attributed to the focal spot, and thereby participates in image formation [4,5]. From
an image reconstruction perspective, the
en face
imaging capability of multiphoton excitation techniques
allows a unique 3D visualization of the vessel wall relative to the blood space that is far more informative
than the multiple perpendicular slices obtained in conventional histological studies. As discussed in this
chapter's Introduction, the microstructural components in the normal and diseased arterial wall are par-
ticularly well suited to multiphoton excitation imaging schemes since they are highly scattering, thereby
compromising high-energy, single-photon excitation studies. In addition, these primary macromolecules
of clinical interest can be detected without exogenous probes, fixation, or tissue sectioning by MEF, HG, or
CARS. Such a multimodal imaging scheme is illustrated in Figure 13.1 [3], where the various components
of the multiphoton emission spectrum of porcine skin are shown. MEF and HG are routinely used in vas-
cular studies, with CARS quickly gaining interest as a means of detecting lipids. Thus, we believe a review
of these three approaches is warranted in the discussion of vascular wall imaging.
13.2.1.1 Multiphoton excitation Fluorescence
MEF is an incoherent process that involves the absorption of two or more photons and the re-emission
of a single photon with a spectral density similar to that in single-photon excitation (see Figure 13.2).
SFG
SHG
CARS
SHG
10
4
10
3
10
2
TPEF
10
1
10
0
400
500
Wavelength (nm)
600
700
FIgurE 13.1
A representative multiphoton emission spectrum of porcine skin generated using two spatially and
temporally overlapped pulsed light sources with λ
1
= 816.7 nm and λ
2
= 1064 nm. The four sharp spectral lines were
resulted from SHG (408.4 and 532 nm), SFG (462 nm), and CARS (663 nm), respectively, whereas the broad spectral
feature on which the four sharp lines are superimposed was from the TPEF. (Reproduced from Jhan, J. W. et al.
2008. Integrated multiple multi-photon imaging and Raman spectroscopy for characterizing structure-constituent
correlation of tissues.
Opt Express
16:16431-16441. With permission of Optical Society of America.)
