depends on the mass of the vibrating atoms. The exact position,
however, is also determined by intra- and intermolecular interactions, both of which alter during dehydration or changes in temperature. Conformational changes of proteins or phase changes of
lipids are visible as abrupt changes in the position and shape of
characteristic absorbance bands. Permeation of solutes in a tissue is
evident as a relative increase in the intensity and area of specific
bands.
The advantage of FTIR is that it is minimally invasive and does
not require labeling, whereas spectra can be recorded in any physical state of a sample (i.e., hydrated, frozen, or dried state). Membrane phase behavior and liquid-to-gel-phase changes during
freezing and drying can be observed by inspecting the position of
the symmetric CH 2 stretching band arising from lipid acyl chains
[3, 9, 10], which is affected by the strength of the van der Waals’
interactions. Changes in protein secondary structure associated
with heat-induced denaturation, intermolecular cross-linking, and
relative contents of α-helical and β-sheet structures can be evaluated
by analyzing the shape of the protein amide bands [11]. Other
cryobiologically relevant physical events that can be captured by
FTIR are the water-to-ice phase transition, which can be derived
from the libration and bending combination band of water [3], and
the glass transition, which can be derived from the OH stretching
band in case of sugar glasses [5, 12]. In principle, different phase
and conformational transitions can be analyzed simultaneously
from the same spectral dataset by analyzing different spectral
regions. Permeation of tissue with protective molecules, and relative solute concentrations, can also be detected by FTIR. Kinetics
of permeation processes can be studied in real time, and mathematical models can be applied to derive diffusion coefficients [6]. Even
permeation of multiple components in mixtures (e.g., vitrification
solutions) can be simultaneously monitored by analyzing characteristic spectral regions of the individual components [8]. FTIR
thus provides a powerful tool for rational design of cryopreservation and freeze-drying methods. In this chapter, spectral data
acquisition and data analysis algorithms for the abovementioned
applications are outlined.
2 Materials
2.1 Fourier
Transform Infrared
Spectrometer
1. A Fourier transform infrared (FTIR) spectrometer (e.g., Frontier spectrometer from PerkinElmer; Nicolet iS5 FTIR spectrometer from Thermo Fisher; ALPHA II FTIR spectrometer
from Bruker), equipped with a narrow band mercury cadmium
telluride (MCT) liquid nitrogen-cooled IR detector (see
Note 1).
332
Willem F. Wolkers and Harrie ¨ tte Oldenhof
however, is also determined by intra- and intermolecular interactions, both of which alter during dehydration or changes in temperature. Conformational changes of proteins or phase changes of
lipids are visible as abrupt changes in the position and shape of
characteristic absorbance bands. Permeation of solutes in a tissue is
evident as a relative increase in the intensity and area of specific
bands.
The advantage of FTIR is that it is minimally invasive and does
not require labeling, whereas spectra can be recorded in any physical state of a sample (i.e., hydrated, frozen, or dried state). Membrane phase behavior and liquid-to-gel-phase changes during
freezing and drying can be observed by inspecting the position of
the symmetric CH 2 stretching band arising from lipid acyl chains
[3, 9, 10], which is affected by the strength of the van der Waals’
interactions. Changes in protein secondary structure associated
with heat-induced denaturation, intermolecular cross-linking, and
relative contents of α-helical and β-sheet structures can be evaluated
by analyzing the shape of the protein amide bands [11]. Other
cryobiologically relevant physical events that can be captured by
FTIR are the water-to-ice phase transition, which can be derived
from the libration and bending combination band of water [3], and
the glass transition, which can be derived from the OH stretching
band in case of sugar glasses [5, 12]. In principle, different phase
and conformational transitions can be analyzed simultaneously
from the same spectral dataset by analyzing different spectral
regions. Permeation of tissue with protective molecules, and relative solute concentrations, can also be detected by FTIR. Kinetics
of permeation processes can be studied in real time, and mathematical models can be applied to derive diffusion coefficients [6]. Even
permeation of multiple components in mixtures (e.g., vitrification
solutions) can be simultaneously monitored by analyzing characteristic spectral regions of the individual components [8]. FTIR
thus provides a powerful tool for rational design of cryopreservation and freeze-drying methods. In this chapter, spectral data
acquisition and data analysis algorithms for the abovementioned
applications are outlined.
2 Materials
2.1 Fourier
Transform Infrared
Spectrometer
1. A Fourier transform infrared (FTIR) spectrometer (e.g., Frontier spectrometer from PerkinElmer; Nicolet iS5 FTIR spectrometer from Thermo Fisher; ALPHA II FTIR spectrometer
from Bruker), equipped with a narrow band mercury cadmium
telluride (MCT) liquid nitrogen-cooled IR detector (see
Note 1).
332
Willem F. Wolkers and Harrie ¨ tte Oldenhof