482
S. Biswas et al.
by the Rayleigh criterion; namely, it was believed that the smallest pixel size at
the sample focal plane should be the diffraction limited spot size and no further
improvement in image quality was possible. Recently, it was realized that this was
not the case. the appropriate pixel size can be understood in two contexts, both
of which are related. First, it is trivial to observe that there is structure in the point
spread function (PSF) of any focusing optic. to access this structure, clearly the
pixel size must be smaller. A suggestion of 5 pixels to sample the entire PSF, implies
that the effective pixel size be approximately five-fold smaller than the Rayleigh
prescribed spot size. this leads directly to a case where the pixels size is approximately 0.5 micron, at the short end of the mid-IR wavelengths. Since this implies
that a 100-fold smaller pixel is optimal compared to most commercial instruments,
a much brighter source was needed. the concepts of proper sampling and smaller
pixels was achieved using a high brightness synchrotron source coupled to a FPA
detector [25]. the major problem in realizing this concept was that the synchrotron
source could illuminate only a small sample area due to its intrinsically smaller
size; hence, either a small number of pixels of the FPA could be used for detection,
or the number of scans had to be significantly increased in order to detect the very
low signal per pixel in a diffused beam. the IRENI (IR Environmental Imaging) at
the Synchrotron Radiation Center in madison, WI was the world’s first IR beamline
to be able to overcome this problem by combining a multiple synchrotron beam
source, along with an FPA detector [28]. this and other systems [29–32] utilize the
advantages of both large-area illumination from a high brightness, stable, broadband synchrotron source and resulting high SNR and spatial resolution along with
the high speed and large area scanning ability of multiple element FPA detectors.
two additional changes were made in order to achieve the optimal imaging for a
given wavelength. First, a high numerical aperture (NA) objective (according to the
Rayleigh criterion) and, second, pixel magnification small enough to be able to accurately scan minute details of the sample.
According to the Rayleigh criterion, the spatial resolution of a microscope is
given by
1.22
2NA
, where NA is given by n sin θ, where n is the refractive index of
the medium and θ is the maximum subtended angle of the objective. As mentioned
previously, commercially-available Ft-IR imaging spectrometers are designed for
a pixel size of ~ 5 × 5 μm to correspond to the mid-band of the mid-IR wavelengths
and a NA of ~ 0.5. the IRENI source on the other hand, used an objective of NA—
0.65, thus increasing its spatial resolution as well. the combination of 12 bright
synchrotron radiation sources in the IRENI set up, is strong enough to enable the
increase of the magnification of the lens to 74x such that, the effective sample-plane
pixel size can be decreased to 0.54 × 0.54 μm
2
. thus both the criteria are satisfied
and this leads to a significant increase in spatial resolution and quality of the image
formed as shown in Fig. 16.4. As a first demonstrative example of the new capabilities, we used it to image, identify and characterize small cell types such as those
in the intralobular stroma in the human breast and the basement membrane in the
human prostate tissue with unprecedented detail.
S. Biswas et al.
by the Rayleigh criterion; namely, it was believed that the smallest pixel size at
the sample focal plane should be the diffraction limited spot size and no further
improvement in image quality was possible. Recently, it was realized that this was
not the case. the appropriate pixel size can be understood in two contexts, both
of which are related. First, it is trivial to observe that there is structure in the point
spread function (PSF) of any focusing optic. to access this structure, clearly the
pixel size must be smaller. A suggestion of 5 pixels to sample the entire PSF, implies
that the effective pixel size be approximately five-fold smaller than the Rayleigh
prescribed spot size. this leads directly to a case where the pixels size is approximately 0.5 micron, at the short end of the mid-IR wavelengths. Since this implies
that a 100-fold smaller pixel is optimal compared to most commercial instruments,
a much brighter source was needed. the concepts of proper sampling and smaller
pixels was achieved using a high brightness synchrotron source coupled to a FPA
detector [25]. the major problem in realizing this concept was that the synchrotron
source could illuminate only a small sample area due to its intrinsically smaller
size; hence, either a small number of pixels of the FPA could be used for detection,
or the number of scans had to be significantly increased in order to detect the very
low signal per pixel in a diffused beam. the IRENI (IR Environmental Imaging) at
the Synchrotron Radiation Center in madison, WI was the world’s first IR beamline
to be able to overcome this problem by combining a multiple synchrotron beam
source, along with an FPA detector [28]. this and other systems [29–32] utilize the
advantages of both large-area illumination from a high brightness, stable, broadband synchrotron source and resulting high SNR and spatial resolution along with
the high speed and large area scanning ability of multiple element FPA detectors.
two additional changes were made in order to achieve the optimal imaging for a
given wavelength. First, a high numerical aperture (NA) objective (according to the
Rayleigh criterion) and, second, pixel magnification small enough to be able to accurately scan minute details of the sample.
According to the Rayleigh criterion, the spatial resolution of a microscope is
given by
1.22
2NA
, where NA is given by n sin θ, where n is the refractive index of
the medium and θ is the maximum subtended angle of the objective. As mentioned
previously, commercially-available Ft-IR imaging spectrometers are designed for
a pixel size of ~ 5 × 5 μm to correspond to the mid-band of the mid-IR wavelengths
and a NA of ~ 0.5. the IRENI source on the other hand, used an objective of NA—
0.65, thus increasing its spatial resolution as well. the combination of 12 bright
synchrotron radiation sources in the IRENI set up, is strong enough to enable the
increase of the magnification of the lens to 74x such that, the effective sample-plane
pixel size can be decreased to 0.54 × 0.54 μm
2
. thus both the criteria are satisfied
and this leads to a significant increase in spatial resolution and quality of the image
formed as shown in Fig. 16.4. As a first demonstrative example of the new capabilities, we used it to image, identify and characterize small cell types such as those
in the intralobular stroma in the human breast and the basement membrane in the
human prostate tissue with unprecedented detail.