Page 132 - Vibrational Spectroscopic Imaging for Biomedical Applications

108 Cha pte r F o u r

FIGURE 4.4 Perkin Elmer ATR imaging accessory.

objectives are achromatic. Since ATR imaging is a reflectance method,
typically one-half of the objective is employed to direct radiation into
the hemisphere, while the other half is employed to collect radiation
internally reflected to the detector (a configuration which is commonly
referred to as an aperture splitting beam splitter). Since only half of the
microscope aperture is employed (sin θ = 0.3), the improvement in
spatial resolution is normally “n/2” instead of “n.” One could employ
the entire aperture of the objective by using a conventional beam
splitter; however, in this case a compromise in signal to noise may be
experienced. In the aperture splitting beam splitter, if 100 photons
were incident on a non-absorbing sample, nearly 100 photons would
be observed at the detector. In the case of a conventional beam splitter,
only 25 photons would be observed. That is, 50 percent of the radi-
ation is lost on the first reflection at the beam splitter and another
50 percent being lost on the second reflection.
Another important consideration for the use of a hemispherical IRE
on a conventional infrared microscope is that the critical angle be met. As
is shown in Fig. 4.1, radiation entering the hemisphere spans a range of
angles that are dictated by the design of the reflecting objective. The most
extreme ray can be found from the numerical aperture of the objective
and the lesser ray can be found by experiment or by contacting the
36
manufacturer. However, because the optical design of the objectives
have been optimized for N.A. = 0.6 the most extreme ray entering the
hemisphere is ~37° and the lesser ray is ~17°. In order for internal
reflection to occur at the IRE/sample interface, radiation entering the
hemisphere must be incident beyond the critical angle given by Eq. (4.5).

n
sinθ = sample (4.5)
−1
n
IRE
Table 4.1 lists several common IRE materials along with the
required critical angle assuming a sample refractive index of 1.5
The data in Table 4.1 demonstrate that only germanium permits
all the radiation to be internally reflected. If ZnSe or diamond were