425
15 FtIR Imaging of tissues: techniques and methods of Analysis
surements to PC12 cells during neuronal differentiation, which was correlated with
phosphorylation using fluorescence microscopy [22]. It has been reported that an
increase in the intensity of the bands at 1080 and between 990 and 970 cm
−1
reflects
sustained elevated phosphorylation of proteins, while the asymmetric phosphate
mode [ν as (Po 2
−
)], which is observed at ca. 1238 cm
−1
, appears to be less sensitive
and less specific to this process [22].
An analysis of FtIR spectra of cells and tissues often includes monitoring of
changes in selected bands ratios. Benedetti and co-workers showed that the ratio
of amide I/amide II bands can serve as a spectral marker for changes in dNA concentration [23]. Since this ratio is equal to unity in human erythrocytes that are
anucleated, its increase can indicate dNA synthesis because of the contribution of
the dNA C = o vibration to the amide I absorbance (spectral features of nucleic acids are discussed in Subsections 2.4 and 2.5). In addition, the ratio of the integrated
absorbances of bands at 1080 (ν s (Po 2
−
)) and 1540 cm
−1
(amide II) was proposed as a
clinical parameter for the evaluation of the degree of malignancy in patients affected
by B-chronic lymphatic leukaemia [23]. A similar observation was reported for rabbit bone marrow fibroblasts transformed by murine sarcoma virus [24]. In the case
of IR imaging of cells, the amide I/ν s (Po 2
−
) ratio gives an insight into the cytoplasm
to nucleus ratio (C/N), since a high protein content is expected to be in the cytoplasm
region of the cell, while nucleic acids are condensed in the cellular nucleus [25].
15.2.2 Lipids
high lipid content mainly arises from plasma membranes and microsomal pellets such as endosomes, secretory vesicles, while only a low lipid concentration is
found in cytoplasm. the most discernible spectral features of lipids or phospholipids originate from the stretching modes of the chain Ch 2 and = Ch groups (2800–
3050 cm
−1
), the ester C = o bond ( ca. 1736 cm
−1
) as well as from the scissoring
vibrations of the long-chain Ch 2 groups ( ca. 1468 cm
−1
), c.f. table 15.1. Biochemical processes, in which lipid metabolism is strongly involved, are mostly recognized by the examination of the high-frequency region. there are usually only four
bands observed from lipids, which are attributed to the asymmetric and symmetric
stretching vibrations of the methyl and methylene groups that are found at ~ 2960
[ν as (Ch 3 )], 2923 [ν as (Ch 2 )], 2873 [ν s (Ch 3 )], and 2850 cm
−1
[ν s (Ch 2 )]. In addition, a
weak band at ca. 3010–3030 cm
−1
is assigned to the Ch stretching vibration of unsaturated fatty acids [ν( = CH)]. The bands of the methylene group are mainly due to
the saturated chains in lipids, whereas the ν(CH 3 ) bands are due to vibrations of the
methyl groups in proteins, lipids, and nucleic acids. the most pronounced spectral
changes indicating alternation of lipid structure and content are found in both the
methylene vibrations [26]. An increase in the ratio of the methylene bands relative
to the ν as (Ch 3 ) band along with the lack of changes in intensity of the ν( = CH) band
shows the presence of predominately long chain saturated fatty acids, whereas an
increase of the ν s (Ch 3 )/ν s (Ch 2 ) ratio is indicative of branched chain fatty acids. If
15 FtIR Imaging of tissues: techniques and methods of Analysis
surements to PC12 cells during neuronal differentiation, which was correlated with
phosphorylation using fluorescence microscopy [22]. It has been reported that an
increase in the intensity of the bands at 1080 and between 990 and 970 cm
−1
reflects
sustained elevated phosphorylation of proteins, while the asymmetric phosphate
mode [ν as (Po 2
−
)], which is observed at ca. 1238 cm
−1
, appears to be less sensitive
and less specific to this process [22].
An analysis of FtIR spectra of cells and tissues often includes monitoring of
changes in selected bands ratios. Benedetti and co-workers showed that the ratio
of amide I/amide II bands can serve as a spectral marker for changes in dNA concentration [23]. Since this ratio is equal to unity in human erythrocytes that are
anucleated, its increase can indicate dNA synthesis because of the contribution of
the dNA C = o vibration to the amide I absorbance (spectral features of nucleic acids are discussed in Subsections 2.4 and 2.5). In addition, the ratio of the integrated
absorbances of bands at 1080 (ν s (Po 2
−
)) and 1540 cm
−1
(amide II) was proposed as a
clinical parameter for the evaluation of the degree of malignancy in patients affected
by B-chronic lymphatic leukaemia [23]. A similar observation was reported for rabbit bone marrow fibroblasts transformed by murine sarcoma virus [24]. In the case
of IR imaging of cells, the amide I/ν s (Po 2
−
) ratio gives an insight into the cytoplasm
to nucleus ratio (C/N), since a high protein content is expected to be in the cytoplasm
region of the cell, while nucleic acids are condensed in the cellular nucleus [25].
15.2.2 Lipids
high lipid content mainly arises from plasma membranes and microsomal pellets such as endosomes, secretory vesicles, while only a low lipid concentration is
found in cytoplasm. the most discernible spectral features of lipids or phospholipids originate from the stretching modes of the chain Ch 2 and = Ch groups (2800–
3050 cm
−1
), the ester C = o bond ( ca. 1736 cm
−1
) as well as from the scissoring
vibrations of the long-chain Ch 2 groups ( ca. 1468 cm
−1
), c.f. table 15.1. Biochemical processes, in which lipid metabolism is strongly involved, are mostly recognized by the examination of the high-frequency region. there are usually only four
bands observed from lipids, which are attributed to the asymmetric and symmetric
stretching vibrations of the methyl and methylene groups that are found at ~ 2960
[ν as (Ch 3 )], 2923 [ν as (Ch 2 )], 2873 [ν s (Ch 3 )], and 2850 cm
−1
[ν s (Ch 2 )]. In addition, a
weak band at ca. 3010–3030 cm
−1
is assigned to the Ch stretching vibration of unsaturated fatty acids [ν( = CH)]. The bands of the methylene group are mainly due to
the saturated chains in lipids, whereas the ν(CH 3 ) bands are due to vibrations of the
methyl groups in proteins, lipids, and nucleic acids. the most pronounced spectral
changes indicating alternation of lipid structure and content are found in both the
methylene vibrations [26]. An increase in the ratio of the methylene bands relative
to the ν as (Ch 3 ) band along with the lack of changes in intensity of the ν( = CH) band
shows the presence of predominately long chain saturated fatty acids, whereas an
increase of the ν s (Ch 3 )/ν s (Ch 2 ) ratio is indicative of branched chain fatty acids. If