485
16 Fourier Transform Infrared (Ft-Ir) Spectroscopic Imaging …
when placing the sample and using a second substrate to sandwich the sample. the
sample, when placed on a single substrate, should be placed such that the substrate
is between the condenser and sample. the sample is exposed to the objective such
that the focal spot can be formed through air and onto the sample. When a substrate
at the top is used, it has been suggested that focusing may be more difficult. hence,
the top substrate should be avoided for both this reason and for the possibility of
causing a cavity induced fringing in the data. By using a novel approach, it was
recently shown that the top substrate can be structured to actually improve data
quality. Kazarian et al. [34, 35] used a hemi-spherical top substrate and demonstrated that the substrate can act as an additional lens to improve magnification in the
optical path. the resulting magnification brings the pixel size closer to the optimal
sampling and, essentially, results in a high-definition type configuration.
16.3.2.2 Transmission-reflection Mode
the transmission-reflection mode (Fig. 16.5b) is attractive as transmission substrates for IR spectroscopy need to be made of salt crystals and are, consequently,
expensive, fragile and often not readily available. the sample usually does not contain an upper substrate and the bottom supporting substrate contains a reflective
(often metallic) coating. the sample preparation is relatively straightforward and
rather similar to that for transmission mode. given the double pass of the beam
through the sample, however, the optimal thickness of the sample is usually smaller
than that for transmission mode. the objective doubles as a condenser in this case.
At times, half the objective is employed to illuminate the sample and half to acquire data. this results in an asymmetric resolution capability and different quality
in the two orthogonal spatial directions. In practical terms, this shortcoming does
not result in an appreciably distorted image, partly because most studies have only
examined the larger pixel case. A more serious effect of the sampling geometry is
the effect on acquired data. the sample acts as a resonant cavity, with light reflected
from the interfaces undergoing multiple reflections and demonstrating the cumulative effect of reflections, scattering and coherent addition. the resulting data are
generally difficult to interpret unless a detailed analysis capturing the physics of the
process is employed. Consequently, there has been much discussion about the utility of this sampling mode and its use for biomedical samples [36–41].
16.3.2.3 Attenuated Total Reflectance (ATR) Mode
As shown in Fig. 16.5c, an AtR crystal is used either as a substrate-objective or as
an overlaid Solid Immersion Lens (SIL) for imaging in this mode. the lens is made
of a material of high refractive index, for example, commonly using germanium,
diamond or Zinc Selenide. In all cases, light is introduced at an angle into the
higher refractive index crystal and transmits to the the lower refractive index sample
interface where it may suffer total internal reflection if the reflction condition is
16 Fourier Transform Infrared (Ft-Ir) Spectroscopic Imaging …
when placing the sample and using a second substrate to sandwich the sample. the
sample, when placed on a single substrate, should be placed such that the substrate
is between the condenser and sample. the sample is exposed to the objective such
that the focal spot can be formed through air and onto the sample. When a substrate
at the top is used, it has been suggested that focusing may be more difficult. hence,
the top substrate should be avoided for both this reason and for the possibility of
causing a cavity induced fringing in the data. By using a novel approach, it was
recently shown that the top substrate can be structured to actually improve data
quality. Kazarian et al. [34, 35] used a hemi-spherical top substrate and demonstrated that the substrate can act as an additional lens to improve magnification in the
optical path. the resulting magnification brings the pixel size closer to the optimal
sampling and, essentially, results in a high-definition type configuration.
16.3.2.2 Transmission-reflection Mode
the transmission-reflection mode (Fig. 16.5b) is attractive as transmission substrates for IR spectroscopy need to be made of salt crystals and are, consequently,
expensive, fragile and often not readily available. the sample usually does not contain an upper substrate and the bottom supporting substrate contains a reflective
(often metallic) coating. the sample preparation is relatively straightforward and
rather similar to that for transmission mode. given the double pass of the beam
through the sample, however, the optimal thickness of the sample is usually smaller
than that for transmission mode. the objective doubles as a condenser in this case.
At times, half the objective is employed to illuminate the sample and half to acquire data. this results in an asymmetric resolution capability and different quality
in the two orthogonal spatial directions. In practical terms, this shortcoming does
not result in an appreciably distorted image, partly because most studies have only
examined the larger pixel case. A more serious effect of the sampling geometry is
the effect on acquired data. the sample acts as a resonant cavity, with light reflected
from the interfaces undergoing multiple reflections and demonstrating the cumulative effect of reflections, scattering and coherent addition. the resulting data are
generally difficult to interpret unless a detailed analysis capturing the physics of the
process is employed. Consequently, there has been much discussion about the utility of this sampling mode and its use for biomedical samples [36–41].
16.3.2.3 Attenuated Total Reflectance (ATR) Mode
As shown in Fig. 16.5c, an AtR crystal is used either as a substrate-objective or as
an overlaid Solid Immersion Lens (SIL) for imaging in this mode. the lens is made
of a material of high refractive index, for example, commonly using germanium,
diamond or Zinc Selenide. In all cases, light is introduced at an angle into the
higher refractive index crystal and transmits to the the lower refractive index sample
interface where it may suffer total internal reflection if the reflction condition is