Biomedical Engineering Reference
In-Depth Information
Strong CARS signal is generated at the C-H and CH
2
stretch vibrational resonance, ω
p
− ω
s
= 2840 cm
−1
,
in lipid-rich cells and extracellular lipid droplets present in atherosclerotic plaques because of their rich
content of these bonds [29]. CARS microscopy is easily amenable to multimodal imaging via the SFG
generated between its pump and Stokes beams as demonstrated in spinal tissues [18]. Addition of a fem-
tosecond pulsed laser enables to visualize collagen with HG and elastin with MEF and therefore cover
the three major components of the arterial wall [29]. It is important to note that elastin and collagen are
also rich in CH
2
bonds and that these components will contribute to the CARS signal when imaging
lipid-rich cells [30]. While this may prevent accurate quantification of these three components, multi-
modal microscopy including CARS, MEF, and SHG is potentially an invaluable tool for image-based
diagnosis [31].
CARS could also prove useful in characterizing water diffusion across the arterial wall. CARS imag-
ing of water diffusion can be achieved using D
2
O as a contrast agent due to the 10-fold increase in CARS
intensity at 3220 cm
−1
of the O-H stretch vibration over that of the O-D stretch vibration. The O-D
stretch vibration on the other hand exhibits two broad peaks at 2385 and 2515 cm
−1
. Single-cell water
dynamics were observed in real time with a CARS microscope by recording intracellular H
2
O/D
2
O
exchanges [23]. The diffusion coefficient across the cell and the membrane permeability were estimated
by fitting a water diffusion model to the spatiotemporal evolution of the CARS signal from a cell during
D
2
O perfusion.
13.3 Arterial Sample Preparations
13.3.1
In Vitro
Excised arteries have been mounted in perfusion chambers that allow the application of intraluminal
pressure, thereby providing a means to systematically study effects of pressure on arterial wall structure
and function
in vitro
[32]. In such studies, the arterial wall is imaged from the adventitia (outer wall);
thus, arterial wall thickness limits the imaging depth, restricting the use of this method to vessels with
small thickness (i.e., mouse arteries). Many studies have imaged excised arterial samples
en face
, either
as (i) aortic ring preparations, allowing cross-sectional imaging from the luminal face to the adven-
titia, or (ii) longitudinally cut vessels, allowing imaging from the luminal surface down to a depth of
~200 μm. Arterial preparations may be imaged
in vitro
with or without chemical fixation. Most studies
use fresh samples maintained in saline or various physiological buffers. In our experience, formalin
or formaldehyde fixation does not alter the collagen HG and elastin MEF signal amplitude. However,
cross-links in tissue components produced by chemical fixation can generate autofluorescent signals not
present in fresh tissue that can interfere with the collagen SHG and elastin autofluorescence signals [33].
Thus, it is prudent to compare imaging of fresh and fixed tissues to assure that no artifactual fluorescent
signals are generated by chemical fixation.
13.3.2
In Vivo
Observation of the dynamic subcellular processes in their normal physiological environment is highly
desirable and can be achieved with
in vivo
microscopy [34-36]. A major complication of high-resolution
in vivo
imaging is physiological tissue motion, which clearly limits the resolution and, to some extent,
signal-to-noise ratio by effectively limiting signal averaging. Physiological motions are linked to cardiac
and respiratory activity and may be enhanced by fluid redistribution during physiological perturbations
[4,35]. These complications have limited the application of multiphoton microscopy to
in vivo
vascular
studies.
One solution to address this issue is to mechanically immobilize thin tissues. In a recent study of ath-
erosclerotic plaques
in vivo
, Yu et al. physically constrained surgically exposed carotid arteries in living
mice between a stainless-steel vessel holder and a cover glass [36]. In this model of atherosclerosis,
