T.P. Wróbel et al.
528
fully find relative composition of all three proteins in each of the image pixels based
on spectra of lyophilized standards. the work was a proof-of-concept for further
development of Ft-IR for diagnostic purposes and was done on section coming
from only one patient.
In 2004 imaging equipment was sufficiently developed that, without using a
synchrotron, a group led by prof. Kazarian studied fragments of aorta using transmission, macro-AtR and micro-AtR imaging [61]. All of these modes of measurements have advantages and limitations. transmission and macro-AtR modes give
a comparable size of the images (with spatial resolution in the 5–20 μm scale),
while micro-ATR gives enhanced resolution of around 2–3 μm. ATR measurements
require much less preparation, since penetration depth of the evanescent wave is in
the 0.2–2 μm range and therefore tissue of any size can be placed and measured,
while for transmission mode a cross-section of approximately 10 μm is required.
the only drawback of AtR measurements is the destructivity of soft tissues, which
is often omitted in discussions, but is still present [1].
the large scale imaging allowed to image the tunica media and cholesteryl esters, claiming a quite uniform distribution of the esters. however, when a microAtR mode was used it became evident that cholesteryl esters form clusters in a
surrounding matrix. generally, a high level of heterogeneity was reported.
the heterogeneity was furthermore confirmed by Wrobel et al. [67] in 2011 with
the Ft-IR transreflection imaging supported by hierarchical Clustering Analysis.
It was shown that by using hCA it was possible to find two atherosclerotic plaque
classes, which after subtraction of their average spectra revealed differences not
only in cholesteryl esters composition but also in that of pure cholesterol. this
structural characterization was followed by work concentrated on protein profile
in atherosclerotic tissue [68]. Again, using hCA and a distance measure based on
correlation coefficient and therefore sensitive to band shape rather than intensity, it
was possible to find a class localized mainly in the plaque. this analysis suggests
that there are specific changes in the secondary structures of proteins inside the lesion.
Functional investigation Functional characterization can be made on many
levels—one of which may be a comparison between samples of aorta with a small
and a developed atherosclerotic plaque (Fig. 17.9). the classes were obtained based
on hCA in 2800–3025 cm
−1
range and the number was decided artificially to represent the morphology of the sample. In the small plaque there are only three classes,
with the red one being assigned to atherosclerotic plaque. In the developed plaque
however, there was a need to have 5 classes in order to fully distinguish the plaque
from the vascular wall. the reason for this is that the blue class in Fig. 17.9c can
be assigned as a fibrous cap, which is more similar to the wall and is not present in
the small plaque. It can be seen that the amount of esters (based on the band around
1735 cm
−1
) is comparable in atherosclerotic classes in both samples, but the difference in the cholesterol band (1055 cm
−1
) quite big.
An interesting functional characterization of atherosclerotic plaques has been
performed with a series of 3 articles by prof. Kazarian group. the first one [64]
528
fully find relative composition of all three proteins in each of the image pixels based
on spectra of lyophilized standards. the work was a proof-of-concept for further
development of Ft-IR for diagnostic purposes and was done on section coming
from only one patient.
In 2004 imaging equipment was sufficiently developed that, without using a
synchrotron, a group led by prof. Kazarian studied fragments of aorta using transmission, macro-AtR and micro-AtR imaging [61]. All of these modes of measurements have advantages and limitations. transmission and macro-AtR modes give
a comparable size of the images (with spatial resolution in the 5–20 μm scale),
while micro-ATR gives enhanced resolution of around 2–3 μm. ATR measurements
require much less preparation, since penetration depth of the evanescent wave is in
the 0.2–2 μm range and therefore tissue of any size can be placed and measured,
while for transmission mode a cross-section of approximately 10 μm is required.
the only drawback of AtR measurements is the destructivity of soft tissues, which
is often omitted in discussions, but is still present [1].
the large scale imaging allowed to image the tunica media and cholesteryl esters, claiming a quite uniform distribution of the esters. however, when a microAtR mode was used it became evident that cholesteryl esters form clusters in a
surrounding matrix. generally, a high level of heterogeneity was reported.
the heterogeneity was furthermore confirmed by Wrobel et al. [67] in 2011 with
the Ft-IR transreflection imaging supported by hierarchical Clustering Analysis.
It was shown that by using hCA it was possible to find two atherosclerotic plaque
classes, which after subtraction of their average spectra revealed differences not
only in cholesteryl esters composition but also in that of pure cholesterol. this
structural characterization was followed by work concentrated on protein profile
in atherosclerotic tissue [68]. Again, using hCA and a distance measure based on
correlation coefficient and therefore sensitive to band shape rather than intensity, it
was possible to find a class localized mainly in the plaque. this analysis suggests
that there are specific changes in the secondary structures of proteins inside the lesion.
Functional investigation Functional characterization can be made on many
levels—one of which may be a comparison between samples of aorta with a small
and a developed atherosclerotic plaque (Fig. 17.9). the classes were obtained based
on hCA in 2800–3025 cm
−1
range and the number was decided artificially to represent the morphology of the sample. In the small plaque there are only three classes,
with the red one being assigned to atherosclerotic plaque. In the developed plaque
however, there was a need to have 5 classes in order to fully distinguish the plaque
from the vascular wall. the reason for this is that the blue class in Fig. 17.9c can
be assigned as a fibrous cap, which is more similar to the wall and is not present in
the small plaque. It can be seen that the amount of esters (based on the band around
1735 cm
−1
) is comparable in atherosclerotic classes in both samples, but the difference in the cholesterol band (1055 cm
−1
) quite big.
An interesting functional characterization of atherosclerotic plaques has been
performed with a series of 3 articles by prof. Kazarian group. the first one [64]