Page 140 - Vibrational Spectroscopic Imaging for Biomedical Applications

116 Cha pte r F o u r

ATR approach, one can limit the optical path length to less than
1 μm. Tisinger has demonstrated that ATR can eliminate artifacts
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associated with edges, specifically for high-contrast edges. The ATR
spectrum collected at the air/protein interface as illustrated in Fig. 4.7
exhibits no such artifacts. Further, the increased spatial resolution of the
ATR method and the limited penetration depth are also useful for the
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study of mineral inclusions in the tissue. The ATR spectrum of
the mineral inclusion illustrated in Fig. 4.8 exhibits features that are
solely calcium oxalate with no protein absorptions. In addition, no
reststrahlen features are present. Last, Fig. 4.7 illustrates a TF spec-
trum collected on an oxalate inclusion. These spectra not only exhibit
scattering and dispersive effects, but poor photometric accuracy as
well. This latter problem is a result of the optical path length through
the sample. The ATR spectrum of the same inclusion exhibits no such
artifacts and is photometrically accurate.
Since it is anticipated that this chapter will be read by histolo-
gists and pathologists, and they appear to be more visually oriented,
the phrase “a picture is worth a 1000 words” is pertinent. Figure 4.7
illustrates infrared images of the same tissue section collected in TF
mode and ATR mode. Infrared images were collected and then a
principal component regression analysis was conducted to deter-
mine how many unique chemical components were present in the
field of view. The different components were then color coded and
plotted as a function of position and intensity. The difference between
the images is quite striking in that the ATR image has better spatial
definition and clarity. The fuzziness in the TF image is a direct result
of the many artifacts discussed above. Spectra extracted from both
images demonstrate, that from a photometric standpoint, the ATR
spectra are clearly superior and could lend themselves to a less prob-
lematic quantitative analysis.
One remaining benefit of the ATR approach is the ability to
detect samples whose sizes are well below the diffraction limit.
Although this can be done with transmission infrared microspec-
troscopy, ATR should prove much better. Patterson has shown that
the detection limit for a micro-ATR measurement is 20 ppm for a
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moderate infrared absorber dissolved in solution. Drawing a sim-
ilar parallel to a small particle in a surrounding matrix, one can
calculate a particle size related to this detection limit. For example,
the spectrum shown in Fig. 4.9 was collected in transmission mode
on a 3-μm diameter polystyrene particle using a 100 × 100 μm aper-
ture. Based on volume arguments the detection limit for the sphere
is ~500 ppm. For the ATR measurement the sampled area is diffrac-
tion limited, so in theory a 6 × 6 μm area would be sampled to a
depth of ~0.7 μm. Using a similar argument, one might expect to be
able to obtain an ATR spectrum of similar quality on a sample as
small as 0.3 μm in diameter at 1000 ppm. Although the 20 ppm
detection limit may not provide sufficient information to identify