Infrared and Raman Spectroscopy and Imaging of Breast Cancer - Vibrational Spectroscopy for Tissue Analysis - page 130

group, and sought to replace or complement traditional histological methods
for brain cancer grading with an automated classification algorithm (more
details are given in the following text).
The most ambitious and comprehensive work to date appears to be that of
Bhargava et al. [58], who sought to establish a very large calibration set using
tissue microarrays (see below).
Size-Scale/resolution issues and instrumentation
Most human cells are on the order of 10 µm in diameter. Mid-infrared wave-
lengths (λ) are 2.5-10 µm. The best attainable (diffraction-limited) spatial
resolution with an infrared microscope is approximately 2λ/3 [65], but this
depends on the design of the instrument and may be limited by the aperture
(for mapping instruments) or the pixel size (for imaging instruments; typically
~5-10 µm). The field of view at the sample depends on the magnifying optics;
some examples for 64 × 64 arrays are 11 × 11 mm [66] (176 µm square pixels),
4 mm × 4 mm (63 µm square pixels) and 500 µm × 500 µm (7.8 µm pixels). The
field of view must be appropriate to the problem at hand. Sometimes indi-
vidual cells, or small groups of cells, are of interest, and high spatial resolu-
tion is required [58]. In other applications, it is necessary to image a larger
tissue sample [66]. The field of view can be effectively increased by using a
motorised sample stage and tiling several images, allowing a large sample to
be imaged at high resolution. A synchrotron source can be used with a point
detector, providing better signal-to-noise ratio with small apertures [65].
Tissue Sampling
All the studies discussed here have used thin sections (4-10 µm) cut from
a larger sample by microtome. The section is then mounted on either an
IR-transparent substrate (e.g., CaF 2 , BaF 2 ) for transmission measurements
or an IR-reflecting substrate (glass slide coated with silver and tin oxide,
or low-E slide ) for reflection-absorption measurements (less common, but
apparently gaining in popularity) [66-69]. One paper discussed tissue micro-
arrays [58] in which core biopsies (500-µm diameter) from many samples are
implanted in a wax block, which is then sliced. This can be thought of as a
high-throughput approach to microscopic tissue imaging.
It is common for parallel sections (another thin slice adjacent to the IR
sample) to be taken and mounted on glass slides for standard histological
analysis, in which contrast is provided by various kinds of staining. When
low-E slides are used, the same sample may be used for IR and visible-light
examination, since these slides are transparent to visible light.
Many studies use samples that have been snap-frozen prior to section-
ing; others use samples that have been archived in wax (paraffin) blocks
(these are more readily available, since hospitals often keep them). Paraffin-
embedded samples are usually de-waxed with xylene prior to analysis.