Biology Reference
In-Depth Information
microscopy provides ultrastructural information of myelin. Histology
allows detection of the demyelinating lesions based on the intensity
difference of the myelin stain in large-view images. Immuno-
fl uorescence with the aid of various antibodies for staining myelin
protein is widely used to examine the morphology and locations of
myelin proteins in nervous tissues. All three methods require the
use of the fi xed slices, complicated sample preparation, and myelin
stain. These procedures inhibit the dynamic study of the process of
myelin damage under the diseased conditions. Clinically, magnetic
resonance imaging (MRI) (
1
) utilizing the diffusive water signal
has been used for detection of demyelinating lesions in multiple
sclerosis patients. It is noninvasive and able to record the demyeli-
nating lesions in the patients for a long time period. Positron emission
tomography (PET) (
2
) is also a noninvasive nuclear imaging technique
which utilizes radio-labeled dye and produces a three-dimensional
image of functional processes in the body. However, these two
methods lack enough resolution for detection of detailed myelin
structure at the single cell level. Therefore, there is a cap between
histological and clinical evaluation. Coherent anti-Stokes Raman
scattering (CARS) microscopy fi lls in the abovementioned niche
by permitting chemically selective imaging of myelin at the resolu-
tion of single cell level. CARS is a nonlinear optical imaging process
and provides inherent 3D submicron resolution. CARS microscopy
allows label-free, high-speed imaging of vibrations of chemical
bonds in the molecules. It can be used for detection of myelin
without any labeling in the fi xed slices, fresh tissue, and even live
animals at the single cell level. With these advances of CARS
microscopy, we have applied CARS microscopy in the mechanistic
study of myelin disruption in various models. In addition to myelin
study, CARS microscopy has been applied to study lipid droplet
(
3, 4
), lipid bilayer (
5, 6
), mouse skin (
7
), and atherosclerotic
lesions (
8, 9
).
In the following sections, we fi rst introduce the principle of
CARS and the components in a CARS microscope. Then, we
describe the procedures using CARS microscopy for imaging of
myelin and myelin changes in various conditions. Finally, we present
the application of CARS microscopy to mechanistic study of myelin
damage in two models.
2. Principle
of CARS
CARS is a nonlinear optical imaging process that is sensitive
to vibrations of molecular chemical bonds (
10, 11
). In a CARS process,
three electromagnetic fi elds produced by lasers at the pump (
w
p
),
Stokes (
w
s
), and probe (
) frequencies interact with molecules in
a medium and generate a new fi eld at the anti-Stokes frequency
w
′
p
wwww
=−+′
. In most experiments, the pump fi eld
E
p
and
(
)
as
p
s
p
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