463
15 FtIR Imaging of tissues: techniques and methods of Analysis
is consistent with observations found in the cervical tissue and other cancerous
tissues, as discussed in Sections 1 and 5.1. here, an increase of dNA content was
confirmed by mgP (methyl green-pyronin) staining for dNA and RNA as well immunohistochemical staining for Ki-67 protein—the marker of proliferative activity
in human cells [152]. the first staining protocol shows the predominant localization of RNA in the rough endoplasmic reticulum and nucleoli in the normal tissue,
while dNA is found in nuclei. Staining with Ki-67 demonstrates that ca. 30 % of
cells are proliferating in the neoplastic tissue. Neoplastic cells typically contain
more nuclear dNA/RNA as a result of genetic and chromosomal interactions [152].
The detailed IR study on liver tissues deglycogenized by α-amylase and digested
by RNase and dNase confirmed these results [153]. Briefly, normal and cirrhotic
liver tissues display predominantly RNA spectral features (the asymmetrical shape
of the 1080 cm
−1
band), whereas the symmetrical dNA band shape dominates in
neoplastic tissue in the region below 1100 cm
−1
.
FtIR imaging has been successfully employed in detecting spectral markers
that characterise the progression of liver fibrinogenesis [154], liver steatosis [155],
and acetaminophen-induced hepatotoxicity [156]. Liver fibrosis is a wound-healing
process, which leads to the formation of a fibrillar extracellular matrix in the liver
as a consequence of injuries, including infections, toxins, and metabolic disorders
[154]. the formation of fibrillar extracellular matrix is associated with the degradation and remodelling of the extracellular matrix, similarly to myocardiopathy
discussed in Sect. 5.3. Although, there are several tests monitoring liver fibrosis, its
early stage cannot be reliably detected. In this experiment, early fibrosis alternations in a rat bile duct ligation (BdL) model were firstly characterized biochemically by elevated levels of ALt, ASt, and bilirubin in serum [154]. these results
suggest the dynamic deterioration of hepatocellular function. Routine h&E staining
shows progressive deposition of extracellular matrix surrounding the newly formed
bile ducts after 2 weeks of BdL. however, mild bile duct proliferation was evident
after the first week. Immunofluorescence staining of collagen and IR spectra from
the liver sections confirmed the h&E results. the collagen band at 1338 cm
−1
illustrated fibrosis distributions throughout the sections. the IR false colour maps
accurately matched the collagen distribution recorded in the immunofluorescence
photomicrograph, even though the spatial resolution of both the methods differ significantly, ca. 10 and 0.5 μm, respectively [154].
the combination of the two imaging techniques, FtIR and toF-SImS, has been
employed to understand mechanisms underlying fatty liver diseases and to identify
markers for diagnosis and prognosis of steatosis [155]. this study has been focused on detection of variations in lipid composition and metabolic abnormalities
in steatotic and non-steatotic hepatocytes. the tissue sections were derived from the
non-tumoral part of liver resection specimens. the comparison of the non-steatotic
and steatotic vesicles clearly shows the absence of amide I and II bands in the
steatotic regions. the IR spectrum of the fatty liver area is typical for lipids and
reveals the presence of the unsaturated fatty acids only in this region of the liver.
the IR false colour maps shows that the steatosis vesicles are surrounded by the
protein-rich tissue and their lipid conformation dramatically differs from structure
15 FtIR Imaging of tissues: techniques and methods of Analysis
is consistent with observations found in the cervical tissue and other cancerous
tissues, as discussed in Sections 1 and 5.1. here, an increase of dNA content was
confirmed by mgP (methyl green-pyronin) staining for dNA and RNA as well immunohistochemical staining for Ki-67 protein—the marker of proliferative activity
in human cells [152]. the first staining protocol shows the predominant localization of RNA in the rough endoplasmic reticulum and nucleoli in the normal tissue,
while dNA is found in nuclei. Staining with Ki-67 demonstrates that ca. 30 % of
cells are proliferating in the neoplastic tissue. Neoplastic cells typically contain
more nuclear dNA/RNA as a result of genetic and chromosomal interactions [152].
The detailed IR study on liver tissues deglycogenized by α-amylase and digested
by RNase and dNase confirmed these results [153]. Briefly, normal and cirrhotic
liver tissues display predominantly RNA spectral features (the asymmetrical shape
of the 1080 cm
−1
band), whereas the symmetrical dNA band shape dominates in
neoplastic tissue in the region below 1100 cm
−1
.
FtIR imaging has been successfully employed in detecting spectral markers
that characterise the progression of liver fibrinogenesis [154], liver steatosis [155],
and acetaminophen-induced hepatotoxicity [156]. Liver fibrosis is a wound-healing
process, which leads to the formation of a fibrillar extracellular matrix in the liver
as a consequence of injuries, including infections, toxins, and metabolic disorders
[154]. the formation of fibrillar extracellular matrix is associated with the degradation and remodelling of the extracellular matrix, similarly to myocardiopathy
discussed in Sect. 5.3. Although, there are several tests monitoring liver fibrosis, its
early stage cannot be reliably detected. In this experiment, early fibrosis alternations in a rat bile duct ligation (BdL) model were firstly characterized biochemically by elevated levels of ALt, ASt, and bilirubin in serum [154]. these results
suggest the dynamic deterioration of hepatocellular function. Routine h&E staining
shows progressive deposition of extracellular matrix surrounding the newly formed
bile ducts after 2 weeks of BdL. however, mild bile duct proliferation was evident
after the first week. Immunofluorescence staining of collagen and IR spectra from
the liver sections confirmed the h&E results. the collagen band at 1338 cm
−1
illustrated fibrosis distributions throughout the sections. the IR false colour maps
accurately matched the collagen distribution recorded in the immunofluorescence
photomicrograph, even though the spatial resolution of both the methods differ significantly, ca. 10 and 0.5 μm, respectively [154].
the combination of the two imaging techniques, FtIR and toF-SImS, has been
employed to understand mechanisms underlying fatty liver diseases and to identify
markers for diagnosis and prognosis of steatosis [155]. this study has been focused on detection of variations in lipid composition and metabolic abnormalities
in steatotic and non-steatotic hepatocytes. the tissue sections were derived from the
non-tumoral part of liver resection specimens. the comparison of the non-steatotic
and steatotic vesicles clearly shows the absence of amide I and II bands in the
steatotic regions. the IR spectrum of the fatty liver area is typical for lipids and
reveals the presence of the unsaturated fatty acids only in this region of the liver.
the IR false colour maps shows that the steatosis vesicles are surrounded by the
protein-rich tissue and their lipid conformation dramatically differs from structure