LDs were also found, however, in an inverted abundance. In conclusion, the formation of LDs composed of unsaturated lipids is proposed as a universal marker of
inflammation.
Raman spectroscopy has been also successfully used to study the process of
apoptosis in various types of cells and under the influence of various factors, e.g.,
chemical compounds [91], including cytotoxic drugs [92, 93] or harmful ultraviolet
radiation [94]. A large part of the reports focuses on investigation of the effect of
anticancer drugs and their chemotherapeutic response in anticancer therapy [92, 95,
96] or is dedicated to the classification and differentiation of cells in apoptosis [97–
99], as well as a detailed description of biochemical changes in this process [100–
103]. Studies on programmed cell death using Raman spectroscopy enabled
defining a number of characteristic changes in the intensity of bands resulting from
changes in the level of bioconstituents. Fazio et al. [104] analyzed the molecular
changes associated with cell aging in the in vitro leukemia model in relation to the
control cell line. Using Raman spectroscopy in correlation with fluorescence
microscopy, markers of the cell aging process and subcellular changes in cellular
organization were found. Okada et al. [105], based on Raman and fluorescence
imaging, studied the cytochrome c alterations in the process of apoptosis. An
extremely interesting work by Brauche et al. [99] raises the problem of distinguishing the processes of apoptosis and necrosis. Although both processes have
different characteristics, the identification of apoptotic and necrotic cells in in vitro
cultures is a challenge. In this study, cells of two different cell lines for early and
advanced apoptosis and necrosis were analyzed using a combination of Raman
spectroscopy and fluorescence microscopy. Progressive apoptosis was diagnosed
by analyzing small band shifts in Raman spectra, e.g., for the amide I band, which
was observed for both cell lines. However, necrotic cells were characterized by the
increased intensity of bands originating from protein vibrations, in contrast to
apoptotic cells in which the reverse effect was observed. In general, the process of
apoptosis was associated with the decrease in the content of proteins and DNA in
cells, which was seen in Raman spectra as the decrease in the intensity of bands at
1007 and 785 cm
–1 , originating from the breathing mode of the phenylalanine ring
and the deformation vibrations of the nucleic acid bases, respectively [96, 98, 102].
It was also shown that the concentration of DNA in the cell nuclei depended on the
stage of apoptosis [99, 100]. The early stages of apoptosis were investigated in EA.
hy926 endothelial cells after activation by Fas ligand and cycloheximide [106].
Both agents are well-known pro-apoptotic inducers triggering programmed death
cell via binding to the membrane receptors and inhibition of protein synthesis,
respectively [107, 108]. The morphological and chemical changes were determined
with the most important conclusion from this work demonstrating that the early
apoptosis resulted in the statistically significant decrease of the protein level inside
all studied cellular structures with the simultaneous increase of the nucleic acids
content. Raman spectroscopy enabled to define these spectroscopic markers of
early-stage apoptosis independently on the activation pathway [106].
6 Small and Large Molecules Investigated by Raman Spectroscopy
175
inflammation.
Raman spectroscopy has been also successfully used to study the process of
apoptosis in various types of cells and under the influence of various factors, e.g.,
chemical compounds [91], including cytotoxic drugs [92, 93] or harmful ultraviolet
radiation [94]. A large part of the reports focuses on investigation of the effect of
anticancer drugs and their chemotherapeutic response in anticancer therapy [92, 95,
96] or is dedicated to the classification and differentiation of cells in apoptosis [97–
99], as well as a detailed description of biochemical changes in this process [100–
103]. Studies on programmed cell death using Raman spectroscopy enabled
defining a number of characteristic changes in the intensity of bands resulting from
changes in the level of bioconstituents. Fazio et al. [104] analyzed the molecular
changes associated with cell aging in the in vitro leukemia model in relation to the
control cell line. Using Raman spectroscopy in correlation with fluorescence
microscopy, markers of the cell aging process and subcellular changes in cellular
organization were found. Okada et al. [105], based on Raman and fluorescence
imaging, studied the cytochrome c alterations in the process of apoptosis. An
extremely interesting work by Brauche et al. [99] raises the problem of distinguishing the processes of apoptosis and necrosis. Although both processes have
different characteristics, the identification of apoptotic and necrotic cells in in vitro
cultures is a challenge. In this study, cells of two different cell lines for early and
advanced apoptosis and necrosis were analyzed using a combination of Raman
spectroscopy and fluorescence microscopy. Progressive apoptosis was diagnosed
by analyzing small band shifts in Raman spectra, e.g., for the amide I band, which
was observed for both cell lines. However, necrotic cells were characterized by the
increased intensity of bands originating from protein vibrations, in contrast to
apoptotic cells in which the reverse effect was observed. In general, the process of
apoptosis was associated with the decrease in the content of proteins and DNA in
cells, which was seen in Raman spectra as the decrease in the intensity of bands at
1007 and 785 cm
–1 , originating from the breathing mode of the phenylalanine ring
and the deformation vibrations of the nucleic acid bases, respectively [96, 98, 102].
It was also shown that the concentration of DNA in the cell nuclei depended on the
stage of apoptosis [99, 100]. The early stages of apoptosis were investigated in EA.
hy926 endothelial cells after activation by Fas ligand and cycloheximide [106].
Both agents are well-known pro-apoptotic inducers triggering programmed death
cell via binding to the membrane receptors and inhibition of protein synthesis,
respectively [107, 108]. The morphological and chemical changes were determined
with the most important conclusion from this work demonstrating that the early
apoptosis resulted in the statistically significant decrease of the protein level inside
all studied cellular structures with the simultaneous increase of the nucleic acids
content. Raman spectroscopy enabled to define these spectroscopic markers of
early-stage apoptosis independently on the activation pathway [106].
6 Small and Large Molecules Investigated by Raman Spectroscopy
175