435
15 FtIR Imaging of tissues: techniques and methods of Analysis
ratio, and consequently requires a large number of scans. this obviously elongates
time of data collection.
A mCt FPA detector is built of up to 128 × 128 elements. Each pixel is 40 × 40
μm
2
, but the pixel projection onto the sample plane is typically 5.5 × 5.5 μm
2
. It is
a common misconception that pixel projection defines spatial resolution of an IR
image. this value does provide one limit to the achievable spatial resolution. the
128 × 128 mCt FPA detector allows simultaneous collection of 16,384 spectra from
all positions within the sampling area of ca. 700 × 700 μm
2
in one tile (one “snapshot”). this method of data collection is called hyperspectral imaging. this dramatically reduces the time needed for data acquisition. Compared to single element or
linear mapping, the advantage of the FPA technique is improved spatial resolution
with comparable S/N ratio. In fact, the FPA system is apertureless, so diffraction
effects are limited through full illumination of each pixel in the imaging array, contrary to mapping microscopes. this results in recording chemical images close to
the diffraction limit along with a very good S/N ratio. this is particularly important
for recording spectra of cells. Lasch and co-workers have demonstrated that the use
of the mapping and imaging techniques affect data collection time, but does not
impact on the spectral features of oral mucosa cells, showing a similar detection
limit for both techniques [27]. the utility of each technique for specific cases and
their respective limitations are illustrated in detail in [53]. modern IR microspectrometers also enable hyperspectral imaging in AtR mode. this technique has been
patented by Agilent. As mentioned above, the use of the germanium IRE provides a
4-fold increase in spatial resolution and sampling area of approx. 70 × 70 μm
2
, since
pixel projection is 1.1 × 1.1 μm
2
. however, Kazarian and Chan have reported that
spatial resolution of micro-AtR imaging is affected by non-uniform illumination of
the Cassegrain objective, resulting in a non-uniform numerical aperture along the
different axes of the image plane [43]. they determined that when applying microAtR imaging to a polyurethane-PmmA interface, the spatial resolution achieved at
1600 cm
−1
with a vertically oriented sample was 4 μm, whereas when the sample
interface was oriented horizontally a 50 % reduction in spatial resolution was obtained compared to the vertical orientation.
15.3.3 Synchrotron Configuration
Conventional FtIR spectrometers are equipped with a black body source (globar)
that gives a much lower density of infrared radiation compared to a synchrotronbased IR source. In fact, an electron storage ring in a synchrotron generates a highly
collimated beam of IR photons produced from a point source, which exhibits a
brilliance of 100–1000 times greater than that of a globar [54, 55]. Such radiation emitted into a narrow range of angles can be delivered through a very small
aperture (down to 3 μm
2
) to the area of interest, on which it becomes diffraction
limited. But the S/N ratio is significantly improved. Since, a globar source illuminates a ca. 100 μm
2
area, the aperture settings are in the range of 20–100 μm
2
for
15 FtIR Imaging of tissues: techniques and methods of Analysis
ratio, and consequently requires a large number of scans. this obviously elongates
time of data collection.
A mCt FPA detector is built of up to 128 × 128 elements. Each pixel is 40 × 40
μm
2
, but the pixel projection onto the sample plane is typically 5.5 × 5.5 μm
2
. It is
a common misconception that pixel projection defines spatial resolution of an IR
image. this value does provide one limit to the achievable spatial resolution. the
128 × 128 mCt FPA detector allows simultaneous collection of 16,384 spectra from
all positions within the sampling area of ca. 700 × 700 μm
2
in one tile (one “snapshot”). this method of data collection is called hyperspectral imaging. this dramatically reduces the time needed for data acquisition. Compared to single element or
linear mapping, the advantage of the FPA technique is improved spatial resolution
with comparable S/N ratio. In fact, the FPA system is apertureless, so diffraction
effects are limited through full illumination of each pixel in the imaging array, contrary to mapping microscopes. this results in recording chemical images close to
the diffraction limit along with a very good S/N ratio. this is particularly important
for recording spectra of cells. Lasch and co-workers have demonstrated that the use
of the mapping and imaging techniques affect data collection time, but does not
impact on the spectral features of oral mucosa cells, showing a similar detection
limit for both techniques [27]. the utility of each technique for specific cases and
their respective limitations are illustrated in detail in [53]. modern IR microspectrometers also enable hyperspectral imaging in AtR mode. this technique has been
patented by Agilent. As mentioned above, the use of the germanium IRE provides a
4-fold increase in spatial resolution and sampling area of approx. 70 × 70 μm
2
, since
pixel projection is 1.1 × 1.1 μm
2
. however, Kazarian and Chan have reported that
spatial resolution of micro-AtR imaging is affected by non-uniform illumination of
the Cassegrain objective, resulting in a non-uniform numerical aperture along the
different axes of the image plane [43]. they determined that when applying microAtR imaging to a polyurethane-PmmA interface, the spatial resolution achieved at
1600 cm
−1
with a vertically oriented sample was 4 μm, whereas when the sample
interface was oriented horizontally a 50 % reduction in spatial resolution was obtained compared to the vertical orientation.
15.3.3 Synchrotron Configuration
Conventional FtIR spectrometers are equipped with a black body source (globar)
that gives a much lower density of infrared radiation compared to a synchrotronbased IR source. In fact, an electron storage ring in a synchrotron generates a highly
collimated beam of IR photons produced from a point source, which exhibits a
brilliance of 100–1000 times greater than that of a globar [54, 55]. Such radiation emitted into a narrow range of angles can be delivered through a very small
aperture (down to 3 μm
2
) to the area of interest, on which it becomes diffraction
limited. But the S/N ratio is significantly improved. Since, a globar source illuminates a ca. 100 μm
2
area, the aperture settings are in the range of 20–100 μm
2
for