Page 126 - Vibrational Spectroscopic Imaging for Biomedical Applications

102 Cha pte r F o u r

samples that are relatively thick. The only limiting requirement is that
the internal reflection element be in intimate contact with the sample.
Another benefit arises from the fact that the volumetric resolution of
the measurement is extremely good. The diffraction-limited volume
of sample illuminated can be estimated as a cone, where the base of
the cone is the diffraction-limited spot size (x,y) of the focused beam
and the height of the cone is the penetration depth d . Based on these
p
considerations, the limiting volume is no greater than 10 femto-liters
over the mid infrared region. One final benefit of immersion is that
the flux of light that can be collected is given by
2
F n 2 sin θ (4.4)
hemisphere
Relative to a measurement conducted in air, 16× more light can
be collected when using a Ge hemisphere. As a result, the optical
conductance of the method is significantly improved. 6,7

4.3 Historical Development
The history of ATR imaging is somewhat fragmented as it draws on
developments in different fields over the last 50 years. The concept of
immersion has long been known and employed by optical designers
to collect more of the available light and focus that light onto a small
area detector. In conventional detection systems the size of the
detector could be reduced by a factor equal to the refractive index if
the detector was placed in optical contact with the plano surface of a
hemisphere. With the detector at the center of curvature, the lens does
not introduce any spherical aberration or coma. In 1976, hemispherical
8
lenses were employed by Chen et al. to observe “surface-electromagnetic
wave enhanced Raman scattering” in an ATR configuration. Mansfield
9
and Kino were the first to employ a solid immersion lens (SIL) to improve
10
the imaging capabilities of a white-light microscope. Using a SIL with a
refractive index of 2, they were able to resolve features with a spatial
frequency of 100 nm at a wavelength of 436 nm. These authors also
proposed the use of a silicon SIL to exploit the methods advantage in
the infrared region. Shortly thereafter, Mansfield et al. demonstrated
these capabilities in a visible imaging microscope outfitted with a
11
CCD detector. From this point on there were numerous publications
that employed solid immersion lenses, both hemispheres and
hyper-hemispheres, to improve the spatial resolution in optical
microscopy, fluorescence microscopy, Raman microspectroscopy, and
optical data storage systems. 12–16
With respect to attenuated total internal reflection, the early
pioneers included Harrick and Fahrenfort who developed the method
to study infrared spectra of organic materials. 17,18 Fahrenfort employed
hemicylinders of alkali halides to demonstrate the ATR method. The
main benefits of the new found technique included the ability to