T.P. Wróbel et al.
518
and calcium hydroxyapatite [86–88]. this method allows the expression of the tissue composition in relative weight percentages/relative weight fractions and provides quantitative information of such things as internal elastic lamina, collagen
fibers, smooth muscle cells, adventitial fat, foam cells/necrotic core, cholesterol and
carotene crystals, as well as calcification [83, 86, 87, 89, 90]. Recently, quantitative
information about the amount of several components in atherosclerotic plaque was
also studied with the use of the unsupervised spectral unmixing algorithm, vertex
component analysis (vCA) [69].
Characteristic bands for the different morphological and biochemical features
present in atherosclerotic plaque with the corresponding compounds assignment
and their modes are presented in table 17.1. Figure 17.6 presents an example of a
chemical image of the cross-sections of a brachiocephalic artery with plaque obtained through the use of the Raman spectroscopy. the results of K-means Clustering (KmC, see Chap. 3.1.1.) represent the morphology of the sample and without
staining, allowed us to recognize such features as remodeled media, heme, internal
elastic lamina/fibrous cap, lipids, calcification and calcium nodule.
the Raman maps were recorded using a WItec confocal CRm alpha 300 Raman
microscope equipped with an air cooled solid state laser operating at 532 nm and a
CCD detector which was cooled to − 82ºC. The laser was coupled to the microscope
via a single mode optical fibre with a diameter of 50 µm. A dry olympus mPLAN
(100/0.90NA) objective was used. Spatial resolution was equal to 1 µm.
the presence of the lipid core is evidenced by bands connected with the cholesterol fatty acid esters and cholesterol. these bands are observed mainly at around
1440, 1670 and 1740 cm
−1
(yellow class—lipids). the bands at 960 cm
−1
and
1072 cm
−1
are characteristic for calcified plaque and are assigned to the symmetric
stretching vibration of phosphate groups and the combination of the antisymmetric
stretching vibration of phosphate groups with the symmetric stretching vibration
of carbonate groups, respectively (calcium nodule class). the gray class represents
the lipid core with calcification features, because the average spectrum of this class
corresponds to hydroxyapatite and lipids. the internal and external elastic laminas contain both elastin and collagen, but with a higher concentration of elastin.
It was shown from Raman studies that elastic lamina is very consistent within and
between coronary artery samples [87]. the Raman spectra of the collagen fiber
and fibrous cap differ from elastic lamina mainly with the absence/decrease of the
bands connected with desmosine and isodesmosine, which are typical for elastin.
moreover we observe the appearance/increase of the band connected with hydroxyproline, which is typical for collagen. In Fig. 17.6, elastic lamina and fibrous cap
are illustrated as a green class. the Raman spectra of tunica media, which is built
mainly from muscle cells, originates mainly from the protein bands feature. the
amide I at 1664 cm
−1
, amide III near 1257 cm
−1
and phenylalanine at 1004 cm
−1
are the main bands of this class. In Fig. 17.6, this class is called remodeled media,
as it corresponds to the pathologically altered tissue. the average spectrum of the
heme class has the strongest marker bands at 745, 1130 and 1580 cm
−1
, which are
connected with the vibrations of the porphyrin ring when the excitation laser wavelength is equal to 532 nm. this class is also interesting to image, as heme features
518
and calcium hydroxyapatite [86–88]. this method allows the expression of the tissue composition in relative weight percentages/relative weight fractions and provides quantitative information of such things as internal elastic lamina, collagen
fibers, smooth muscle cells, adventitial fat, foam cells/necrotic core, cholesterol and
carotene crystals, as well as calcification [83, 86, 87, 89, 90]. Recently, quantitative
information about the amount of several components in atherosclerotic plaque was
also studied with the use of the unsupervised spectral unmixing algorithm, vertex
component analysis (vCA) [69].
Characteristic bands for the different morphological and biochemical features
present in atherosclerotic plaque with the corresponding compounds assignment
and their modes are presented in table 17.1. Figure 17.6 presents an example of a
chemical image of the cross-sections of a brachiocephalic artery with plaque obtained through the use of the Raman spectroscopy. the results of K-means Clustering (KmC, see Chap. 3.1.1.) represent the morphology of the sample and without
staining, allowed us to recognize such features as remodeled media, heme, internal
elastic lamina/fibrous cap, lipids, calcification and calcium nodule.
the Raman maps were recorded using a WItec confocal CRm alpha 300 Raman
microscope equipped with an air cooled solid state laser operating at 532 nm and a
CCD detector which was cooled to − 82ºC. The laser was coupled to the microscope
via a single mode optical fibre with a diameter of 50 µm. A dry olympus mPLAN
(100/0.90NA) objective was used. Spatial resolution was equal to 1 µm.
the presence of the lipid core is evidenced by bands connected with the cholesterol fatty acid esters and cholesterol. these bands are observed mainly at around
1440, 1670 and 1740 cm
−1
(yellow class—lipids). the bands at 960 cm
−1
and
1072 cm
−1
are characteristic for calcified plaque and are assigned to the symmetric
stretching vibration of phosphate groups and the combination of the antisymmetric
stretching vibration of phosphate groups with the symmetric stretching vibration
of carbonate groups, respectively (calcium nodule class). the gray class represents
the lipid core with calcification features, because the average spectrum of this class
corresponds to hydroxyapatite and lipids. the internal and external elastic laminas contain both elastin and collagen, but with a higher concentration of elastin.
It was shown from Raman studies that elastic lamina is very consistent within and
between coronary artery samples [87]. the Raman spectra of the collagen fiber
and fibrous cap differ from elastic lamina mainly with the absence/decrease of the
bands connected with desmosine and isodesmosine, which are typical for elastin.
moreover we observe the appearance/increase of the band connected with hydroxyproline, which is typical for collagen. In Fig. 17.6, elastic lamina and fibrous cap
are illustrated as a green class. the Raman spectra of tunica media, which is built
mainly from muscle cells, originates mainly from the protein bands feature. the
amide I at 1664 cm
−1
, amide III near 1257 cm
−1
and phenylalanine at 1004 cm
−1
are the main bands of this class. In Fig. 17.6, this class is called remodeled media,
as it corresponds to the pathologically altered tissue. the average spectrum of the
heme class has the strongest marker bands at 745, 1130 and 1580 cm
−1
, which are
connected with the vibrations of the porphyrin ring when the excitation laser wavelength is equal to 532 nm. this class is also interesting to image, as heme features