432
K. malek et al.
Commercially available infrared microscopes are also designed in two configurations, called infinity correction and non-infinity correction. the infinity corrected
design provides flexibility in choice of additional optical elements (objectives, filters), on which the beam is collimated. this system has the focal point at infinity
for sharp focus in both visible and infrared images by collimating the two beams
within the microscope. the non-infinity corrected microscopes are limited in terms
of additional optics, which consequently affects the quality of focus and sharpness
of the image. Among manufactures of IR microscopes, varian (now Agilent) and
thermo Fisher produce the infinity-corrected microscopes, whereas Bruker and
PerkinElmer employ the non-infinity corrected design.
FtIR spectra of cells and tissues can be recorded in three modes including transmission, transflection (or transmission/reflection), and AtR. Since the best S/N is
found for the transmission mode, this technique is the most common for single cell
analysis. For a transmission measurement the sample must be placed on a substrate
made of a material permeable for IR light such as non-hygroscopic CaF 2 , BaF 2 or
CsI windows. to avoid high absorption, the thickness of a sample should be less
than 10 μm. These IR substrates create chromatic aberration, which results in a
non-linear wavelength-dependent focus increasing in the low-frequency spectral
region but this problem is minimized in windows with a thickness less than 1 mm.
For tissue measurements Ag/Sno 2 coated infrared reflective slides (Kevley technologies) are often employed. In this case the measurements are performed in transflection mode, because the beam passes through the sample and reflects off the IR
reflective layer. the beam thus passes through the section twice, and consequently
a suitable thickness of the tissue samples is usually ca. 4 μm. The improved absorbance signal, especially for very thin samples such as tissue sections, the absence of
chromatic aberration along with the low cost of the reflective substrates in the comparison to the permeable IR widows make the transflection technique a useful tool
for IR microspectroscopy of cell, tissues, and biofilms. however, Filik et al. and
Bassan et al. reported that transflection IR spectra can be strongly affected by the
electric field standing wave (EFSW) effect [10, 44]. the EFSW within multilayer
systems results in an inhomogeneous field due to focusing and interference in both
transmission and reflection processes. this leads to the non-linear relationship between absorbance and the sample thickness, which distorts the spectra by raising the
absorbance of the amide I and amide II region relative to the high-frequency range
above ca. 2800 cm
−1
. Even for differences in thickness of 0.1 μm that are common
in morphology of cells and tissues prepared for IR imaging, significant changes in
shapes and ratios of IR bands can occur as a result of this effect. therefore, transflection spectra of biological material should be carefully examined, to confirm that
the observed spectral changes result from the biochemical status of the cell or tissue
under investigation and not from spurious artefacts generated by the transflection
mode of the measurement. this can be overcome to some degree in the case of tissues by having very even tissue sections and by using second derivatives, which are
sensitive to shifts in band position and do not solely rely on direct absorbance [10].
Since the numerical aperture NA is defined as NA = n sinθ, where n is the refractive index of the medium, in which the optics are surrounded and θ is the half-angle
K. malek et al.
Commercially available infrared microscopes are also designed in two configurations, called infinity correction and non-infinity correction. the infinity corrected
design provides flexibility in choice of additional optical elements (objectives, filters), on which the beam is collimated. this system has the focal point at infinity
for sharp focus in both visible and infrared images by collimating the two beams
within the microscope. the non-infinity corrected microscopes are limited in terms
of additional optics, which consequently affects the quality of focus and sharpness
of the image. Among manufactures of IR microscopes, varian (now Agilent) and
thermo Fisher produce the infinity-corrected microscopes, whereas Bruker and
PerkinElmer employ the non-infinity corrected design.
FtIR spectra of cells and tissues can be recorded in three modes including transmission, transflection (or transmission/reflection), and AtR. Since the best S/N is
found for the transmission mode, this technique is the most common for single cell
analysis. For a transmission measurement the sample must be placed on a substrate
made of a material permeable for IR light such as non-hygroscopic CaF 2 , BaF 2 or
CsI windows. to avoid high absorption, the thickness of a sample should be less
than 10 μm. These IR substrates create chromatic aberration, which results in a
non-linear wavelength-dependent focus increasing in the low-frequency spectral
region but this problem is minimized in windows with a thickness less than 1 mm.
For tissue measurements Ag/Sno 2 coated infrared reflective slides (Kevley technologies) are often employed. In this case the measurements are performed in transflection mode, because the beam passes through the sample and reflects off the IR
reflective layer. the beam thus passes through the section twice, and consequently
a suitable thickness of the tissue samples is usually ca. 4 μm. The improved absorbance signal, especially for very thin samples such as tissue sections, the absence of
chromatic aberration along with the low cost of the reflective substrates in the comparison to the permeable IR widows make the transflection technique a useful tool
for IR microspectroscopy of cell, tissues, and biofilms. however, Filik et al. and
Bassan et al. reported that transflection IR spectra can be strongly affected by the
electric field standing wave (EFSW) effect [10, 44]. the EFSW within multilayer
systems results in an inhomogeneous field due to focusing and interference in both
transmission and reflection processes. this leads to the non-linear relationship between absorbance and the sample thickness, which distorts the spectra by raising the
absorbance of the amide I and amide II region relative to the high-frequency range
above ca. 2800 cm
−1
. Even for differences in thickness of 0.1 μm that are common
in morphology of cells and tissues prepared for IR imaging, significant changes in
shapes and ratios of IR bands can occur as a result of this effect. therefore, transflection spectra of biological material should be carefully examined, to confirm that
the observed spectral changes result from the biochemical status of the cell or tissue
under investigation and not from spurious artefacts generated by the transflection
mode of the measurement. this can be overcome to some degree in the case of tissues by having very even tissue sections and by using second derivatives, which are
sensitive to shifts in band position and do not solely rely on direct absorbance [10].
Since the numerical aperture NA is defined as NA = n sinθ, where n is the refractive index of the medium, in which the optics are surrounded and θ is the half-angle