486
S. Biswas et al.
matched. An exponentially decreasing evanescent wave is set up at the interface,
which penetrates into the sample. the depth of penetration of the evanescent wave
depends on the incident angle, refractive indices of the crystal and the sample, and
the radiation wavelength. In this case, therefore, the sample does not directly interact with the IR beam, rather it’s layers closest to the crystal surface interact with the
decaying evanescent wave in a depth weighted manner (19). For commonly used
AtR lenses, the depth of penetration is typically of the order of the wavelength of
light in the IR. these properties can be used to record data from both thick samples
as well as from depth-resolved areas of a sample.
Some of the drawbacks of imaging by transmission or transflectance modes
have been solved using the AtR mode: first, the problem of water absorption can
be mitigated due to the shallow penetration depth and live, fresh or frozen tissues
can be imaged. Second, the NA of the transmission mode characteristically ranges
from 0.4 to 0.8. hence, the smallest beam spot size is approximately equal to the
wavelength. given the high refractive index of the SIL, upto a 4-fold smaller beam
spot size can be attained, making it possible to study small cells like endothelial
cells or myoepithelial cells, which may be important for diagnosing cancer. As a
demonstrative example, using AtR Ft-IR imaging (spatial resolution better than the
pixel size of 1.56 × 1.56 µm), finer structure in breast tissue could be imaged that
was not visible in the other sampling modes as shown in Fig. 16.6. one of the major
advantages of this mode is that sample preparation can be minimal, because processes like sectioning, fixation, or staining can be avoided. AtR imaging hence can
be used in some biomedical applications where the transmission mode would fail
to provide sufficient light intensity for measurements, for cases where finer structure needs to be imaged and for those situations in which preparing thin sections of
samples is difficult. due to sub-wavelength coupling, the approach may also allow
for the analysis samples with strongly scattering profiles. For example, the analysis
of kidney stones without being affected by strong scattering from specular reflection, Christiansen effect and Restsrahlen effects unlike the transmission mode [42].
16.3.3 Fundamental Theoretical Understanding of FT-IR
Microspectroscopy and Imaging
one of the most exciting developments in recent years has been the evolution of theoretical understanding in microscopy and image formation in the mid-IR [43, 44].
the most common theoretical treatment for IR microscopy had been to utilize ray or
geometric optics. While these approaches were useful in designing instruments and
generally predicting the flux and spot size at the focal plane, they are insufficient in
describing optical phenomena when the wavelength is approximately equal to sample dimensions. due to strong absorption, samples are typically microns thick in the
mid-IR. many biological samples are heterogeneous on the micron scale as well.
thus, there are strong effects in the recorded spectrum that arise from the coupling
of micron-wavelength light with micron-dimension samples. the effects include
S. Biswas et al.
matched. An exponentially decreasing evanescent wave is set up at the interface,
which penetrates into the sample. the depth of penetration of the evanescent wave
depends on the incident angle, refractive indices of the crystal and the sample, and
the radiation wavelength. In this case, therefore, the sample does not directly interact with the IR beam, rather it’s layers closest to the crystal surface interact with the
decaying evanescent wave in a depth weighted manner (19). For commonly used
AtR lenses, the depth of penetration is typically of the order of the wavelength of
light in the IR. these properties can be used to record data from both thick samples
as well as from depth-resolved areas of a sample.
Some of the drawbacks of imaging by transmission or transflectance modes
have been solved using the AtR mode: first, the problem of water absorption can
be mitigated due to the shallow penetration depth and live, fresh or frozen tissues
can be imaged. Second, the NA of the transmission mode characteristically ranges
from 0.4 to 0.8. hence, the smallest beam spot size is approximately equal to the
wavelength. given the high refractive index of the SIL, upto a 4-fold smaller beam
spot size can be attained, making it possible to study small cells like endothelial
cells or myoepithelial cells, which may be important for diagnosing cancer. As a
demonstrative example, using AtR Ft-IR imaging (spatial resolution better than the
pixel size of 1.56 × 1.56 µm), finer structure in breast tissue could be imaged that
was not visible in the other sampling modes as shown in Fig. 16.6. one of the major
advantages of this mode is that sample preparation can be minimal, because processes like sectioning, fixation, or staining can be avoided. AtR imaging hence can
be used in some biomedical applications where the transmission mode would fail
to provide sufficient light intensity for measurements, for cases where finer structure needs to be imaged and for those situations in which preparing thin sections of
samples is difficult. due to sub-wavelength coupling, the approach may also allow
for the analysis samples with strongly scattering profiles. For example, the analysis
of kidney stones without being affected by strong scattering from specular reflection, Christiansen effect and Restsrahlen effects unlike the transmission mode [42].
16.3.3 Fundamental Theoretical Understanding of FT-IR
Microspectroscopy and Imaging
one of the most exciting developments in recent years has been the evolution of theoretical understanding in microscopy and image formation in the mid-IR [43, 44].
the most common theoretical treatment for IR microscopy had been to utilize ray or
geometric optics. While these approaches were useful in designing instruments and
generally predicting the flux and spot size at the focal plane, they are insufficient in
describing optical phenomena when the wavelength is approximately equal to sample dimensions. due to strong absorption, samples are typically microns thick in the
mid-IR. many biological samples are heterogeneous on the micron scale as well.
thus, there are strong effects in the recorded spectrum that arise from the coupling
of micron-wavelength light with micron-dimension samples. the effects include