430
K. malek et al.
broad in dehydrated cells and fixed tissues and are often overlapped by glycogen,
collagen, phosphorylated proteins and phosphodiester groups associated with lipids
and fatty acids. A shoulder feature on the amide I mode appearing at 1712 cm
−1
in
dehydrated cells and at 1716 cm
−1
in hydrated cells is assigned to in-plane base
C = C and C = o double bonds stretching from dNA. A band at 1050 cm
−1
assigned
to the C-o stretching vibration from deoxyribose shows a dramatic increase in intensity as a function of increasing hydration. the band at 967 cm
−1
is assigned to
a C-C stretching vibration based on its similarity to the 950 cm
−1
band observed
diethyl phosphate anion [39]. this band also appears sharper and more intense in
the hydrated state.
It is hypothesized that as the dNA transforms from the B-dNA conformation
to the A-dNA form the overall molar extinction coefficient decreases because of
the disordered alignment of the individual molecular dipoles in the phosphodiester
backbone. this has the effect of broadening the asymmetric and symmetric phosphodiester stretching modes to the point where they are overlapped by bands from
other molecules. In summary, it is important to control the humidity when working
with air-dried cells. If one wants to visualize the dNA more clearly then the cells
should be hydrated or a fixative that preserves the B-dNA form needs to be applied.
Any type of fixation will result in the formation of A-dNA, broadening the dNA
bands to the point where they are not perceptible against the superposition of other
bands in the region.
15.3 Instrumentation for FTIR Imaging
15.3.1 FTIR Microscope Configurations
the coupling of an FtIR spectrometer with a Cassegrain objective microscope has
introduced a powerful tool for simultaneous analysis of the chemical structure and
spatial distribution in heterogeneous materials of micron size (~ 2.5–25 μm). This
has opened up the possibility of diagnostics of biochemical changes to the level
of the single cell. IR microscopes are designed to operate in either transmission or
reflection mode. In addition, the microscope can be equipped with a micro-AtR
accessory (usually germanium crystal) allowing collection of attenuated total reflection IR spectra (AtR FtIR). transmission or transflection modes are the most
popular modes used to investigate cells and tissues. to collect FtIR spectra in
transmission mode, the IR beam from the interferometer passes to the Cassegrain
condenser—a two mirror optic. there, the beam is focused on a sample placed on
an x, y, z motion stage and transmitted through the sample to the Cassegrain objective, and next to an adjustable aperture. After that, the radiation is focused onto a
detector, which records the spectrum from the sample area limited by the aperture.
the region of interest is imaged by using a CCd camera. Figure 15.4 illustrates a
simplified scheme of the IR microscope.
K. malek et al.
broad in dehydrated cells and fixed tissues and are often overlapped by glycogen,
collagen, phosphorylated proteins and phosphodiester groups associated with lipids
and fatty acids. A shoulder feature on the amide I mode appearing at 1712 cm
−1
in
dehydrated cells and at 1716 cm
−1
in hydrated cells is assigned to in-plane base
C = C and C = o double bonds stretching from dNA. A band at 1050 cm
−1
assigned
to the C-o stretching vibration from deoxyribose shows a dramatic increase in intensity as a function of increasing hydration. the band at 967 cm
−1
is assigned to
a C-C stretching vibration based on its similarity to the 950 cm
−1
band observed
diethyl phosphate anion [39]. this band also appears sharper and more intense in
the hydrated state.
It is hypothesized that as the dNA transforms from the B-dNA conformation
to the A-dNA form the overall molar extinction coefficient decreases because of
the disordered alignment of the individual molecular dipoles in the phosphodiester
backbone. this has the effect of broadening the asymmetric and symmetric phosphodiester stretching modes to the point where they are overlapped by bands from
other molecules. In summary, it is important to control the humidity when working
with air-dried cells. If one wants to visualize the dNA more clearly then the cells
should be hydrated or a fixative that preserves the B-dNA form needs to be applied.
Any type of fixation will result in the formation of A-dNA, broadening the dNA
bands to the point where they are not perceptible against the superposition of other
bands in the region.
15.3 Instrumentation for FTIR Imaging
15.3.1 FTIR Microscope Configurations
the coupling of an FtIR spectrometer with a Cassegrain objective microscope has
introduced a powerful tool for simultaneous analysis of the chemical structure and
spatial distribution in heterogeneous materials of micron size (~ 2.5–25 μm). This
has opened up the possibility of diagnostics of biochemical changes to the level
of the single cell. IR microscopes are designed to operate in either transmission or
reflection mode. In addition, the microscope can be equipped with a micro-AtR
accessory (usually germanium crystal) allowing collection of attenuated total reflection IR spectra (AtR FtIR). transmission or transflection modes are the most
popular modes used to investigate cells and tissues. to collect FtIR spectra in
transmission mode, the IR beam from the interferometer passes to the Cassegrain
condenser—a two mirror optic. there, the beam is focused on a sample placed on
an x, y, z motion stage and transmitted through the sample to the Cassegrain objective, and next to an adjustable aperture. After that, the radiation is focused onto a
detector, which records the spectrum from the sample area limited by the aperture.
the region of interest is imaged by using a CCd camera. Figure 15.4 illustrates a
simplified scheme of the IR microscope.