standard offered for each application. In general, the tissue slices are placed directly
onto CaF 2 or glass windows that possess a simple or well-defined Raman spectrum
beyond the studied spectral region. An optimal option is to study raw, not fixed
tissue samples, since most fixation agents contribute to a Raman spectrum. This is
achievable in vivo using Raman fiber optic probes using fast-data collecting
techniques such as coherent anti-Stokes Raman spectroscopy [162] or simply for
tissues not sensitive to external conditions, i.e., for aorta cross-sections [72]. Tissue
cross sections are usually preserved because of long time of spectral acquisition. To
extend storage of tissue samples, two common methods can be employed, i.e.,
chemical or non-chemical fixation using, for example, air-drying. Among chemical
methods, the most frequently used protocol is fixation in the formalin solution.
Hydrated formalin cross-links the primary and secondary amine groups of proteins
preventing their autolysis and stabilizing a tissue structure. Effects of formalin
fixation on Raman spectra of mammalian animal tissues and cells have been previously studied and summarized [163].
The choice of a laser excitation line is another crucial element of Raman imaging
of tissues. Most of the research is performed with using near-infrared lasers, mainly
with a 785-nm line, due to huge autofluorescence arising from connective tissues
when they are irradiated by visible light sources. In case of tissues containing blood,
a NIR laser excitation is required to omit the contribution of a resonance Raman
effect of heme moieties which strongly overlaps Raman signal of other biocomponents. However, one must know that high lateral resolution is achieved in
microscopic Raman imaging when UV and Vis lasers are employed.
In recent decades, Raman spectroscopy has been successfully applied to show
tissue alterations occurring due to development of lifestyle diseases and various
types of cancers [71, 161, 164, 165]. A proper Raman analysis of these changes
associated with a disease requires to understand biochemistry of healthy tissue. In a
review article published by us [166], the chemical composition of tissue
cross-sections and homogenates of various organs, inter alia, kidney, brain, liver,
and aorta, were discussed in terms of their characteristic Raman signatures. As was
above mentioned, the vascular endothelium status is a determinant of the condition
of the cardiovascular system, and its dysfunction is at the root of diseases of
affluence. An examination of the endothelium in ex vivo cross-sections of the
murine aorta has been performed for animal models of diabetes type 2 [74],
hypertension [60], and cancer metastasis [72]. Common features of endothelial
dysfunction in the three pathologies are variations in an overall amount of proteins
and/or lipids in this layer of the vessel. In addition, confocal Raman and AFM
imaging clearly show the formation of lipid rafts’ clusters on the endothelium
surface in the diabetic aorta (Fig. 6.9i). Raman spectroscopy was also used to study
a blood vessel wall to characterize the chemical composition of atherosclerotic
plaque in mice fed with different diets [167]. For example, in ApoE/LDLR
−/− mice
on low-carbonate–high-protein (LCHP) diet within brachiocephalic arteries, various
components in the plague deposit were identified without labelling, including
cholesterol esters and inorganic calcium salts (Fig. 6.9ii). A high lipid-to-protein
ratio and an increased content of cholesterol were also found in a murine brain
184
K. Czamara et al.
onto CaF 2 or glass windows that possess a simple or well-defined Raman spectrum
beyond the studied spectral region. An optimal option is to study raw, not fixed
tissue samples, since most fixation agents contribute to a Raman spectrum. This is
achievable in vivo using Raman fiber optic probes using fast-data collecting
techniques such as coherent anti-Stokes Raman spectroscopy [162] or simply for
tissues not sensitive to external conditions, i.e., for aorta cross-sections [72]. Tissue
cross sections are usually preserved because of long time of spectral acquisition. To
extend storage of tissue samples, two common methods can be employed, i.e.,
chemical or non-chemical fixation using, for example, air-drying. Among chemical
methods, the most frequently used protocol is fixation in the formalin solution.
Hydrated formalin cross-links the primary and secondary amine groups of proteins
preventing their autolysis and stabilizing a tissue structure. Effects of formalin
fixation on Raman spectra of mammalian animal tissues and cells have been previously studied and summarized [163].
The choice of a laser excitation line is another crucial element of Raman imaging
of tissues. Most of the research is performed with using near-infrared lasers, mainly
with a 785-nm line, due to huge autofluorescence arising from connective tissues
when they are irradiated by visible light sources. In case of tissues containing blood,
a NIR laser excitation is required to omit the contribution of a resonance Raman
effect of heme moieties which strongly overlaps Raman signal of other biocomponents. However, one must know that high lateral resolution is achieved in
microscopic Raman imaging when UV and Vis lasers are employed.
In recent decades, Raman spectroscopy has been successfully applied to show
tissue alterations occurring due to development of lifestyle diseases and various
types of cancers [71, 161, 164, 165]. A proper Raman analysis of these changes
associated with a disease requires to understand biochemistry of healthy tissue. In a
review article published by us [166], the chemical composition of tissue
cross-sections and homogenates of various organs, inter alia, kidney, brain, liver,
and aorta, were discussed in terms of their characteristic Raman signatures. As was
above mentioned, the vascular endothelium status is a determinant of the condition
of the cardiovascular system, and its dysfunction is at the root of diseases of
affluence. An examination of the endothelium in ex vivo cross-sections of the
murine aorta has been performed for animal models of diabetes type 2 [74],
hypertension [60], and cancer metastasis [72]. Common features of endothelial
dysfunction in the three pathologies are variations in an overall amount of proteins
and/or lipids in this layer of the vessel. In addition, confocal Raman and AFM
imaging clearly show the formation of lipid rafts’ clusters on the endothelium
surface in the diabetic aorta (Fig. 6.9i). Raman spectroscopy was also used to study
a blood vessel wall to characterize the chemical composition of atherosclerotic
plaque in mice fed with different diets [167]. For example, in ApoE/LDLR
−/− mice
on low-carbonate–high-protein (LCHP) diet within brachiocephalic arteries, various
components in the plague deposit were identified without labelling, including
cholesterol esters and inorganic calcium salts (Fig. 6.9ii). A high lipid-to-protein
ratio and an increased content of cholesterol were also found in a murine brain
184
K. Czamara et al.