Page 127 - Vibrational Spectroscopic Imaging for Biomedical Applications

Evanescent W ave Imaging 103

analyze materials with little or no sample preparation and the ability
to analyze highly absorbing materials. With ATR, the only sample
requirement is that it must be placed into optical (intimate) contact
with the IRE. In addition, the limited depth to which the evanescent
beam penetrated the sample meant that spectra of strongly absorbing
materials could be obtained without total absorption of the infrared
radiation at a particular wavelength. Microscopic ATR methods did
not become available until 1991 when Harrick developed the Split-pea
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infrared microscope and Spectra-Tech independently developed a
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specialized ATR objective for their IRPLAN microscope. The Split-pea
employed a germanium or silicon hemisphere with a beveled tip to
improve contact with the sample and the appearance of the IRE led to
the name of the device. The Spectra-Tech ATR objective employed a
zinc selenide IRE and later a diamond IRE so that the user could
observe the sample in white light prior to conducting an ATR analysis.
Perkin Elmer later developed a dropdown accessory for their micro-
scope, which was based on a germanium hemisphere possessing a
beveled tip. The user simply aligns the sample in white light viewing
mode and then lowers the IRE onto the sample for subsequent infrared
analysis. An added benefit of these devices stems from the fact that the
pressure applied to a given sample is the force divided by the area.
Since the contact area is on the order of 100 to 200 μm for each device,
the pressure and therefore the contact of the IRE with the sample
increased tremendously as compared to a macro sampling accessory.
At that time, the major focus of the devices was on the ability to collect
infrared spectra from intractable samples and not necessarily the
improvement in spatial resolution.
The first reports to study the improved spatial resolution of an
infrared ATR measurement using a germanium IRE was that by
Nakano and Kawata. 21,22 The authors built a specialized evanescent
wave microscope that incorporated a confocal aperture for both the
source and primary image of the sample to spatially isolate the
sample of interest (Fig. 4.2). The hemisphere with attached sample
was translated beneath the microscope using a piezoelectrically
controlled stage. As shown in Fig. 4.2, when the hemisphere is on
axis, rays enter the hemisphere normal to its surface and, as such,
are focused at the center of the plano surface. Moving the hemi-
sphere off-axis to either side, the rays enter at a slight angle, are
refracted, and come to a focus at off-axis positions, thereby allowing
different sample points to be interrogated. The authors demonstrated
an improvement in spatial resolution equal to the refractive index of
germanium (4×) and the ability to scan over an area of approximately
100 μm. The limited scan length was the result of spherical aberrations
introduced by scanning the hemisphere off-axis. In 1995, Esaki et al.
employed a chevron-shaped internal reflection element (Fig. 4.2) on a
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conventional microscope. Esaki et al. demonstrated the ability to
obtain ATR maps as large as 400 × 400 μm. However, since a hemisphere