Page 84 - Vibrational Spectroscopic Imaging for Biomedical Applications

60 Cha pte r T h ree

the application of FTIR to cell analysis for diagnostic or imaging pur-
poses reveals that cells had generally been prepared by direct culture
upon IR transparent substrates, then removal from culture medium
1–6
and air-drying. However, the air-drying process causes delocaliza-
tion of biomolecules as a result of large surface tension forces associ-
ated with the passing water-air interface. Other researchers in the
field had prepared cells by removing them from culture medium and
7
8
then centrifugation, drying under nitrogen gas or cytospinning 9
with the view of minimizing the effects of these surface tension forces
by increasing the rate of dehydration.
The removal of cells from pH buffered growth medium and sub-
sequent air-drying can influence the osmotic pressure within these
cells, resulting in cell shrinkage or swelling with the latter resulting in
membrane rupture and leaching of intercellular components. In addi-
tion, drying of living cells can initiate autolytic processes whereby
intracellular enzymes contained within lysosomes cause denaturing
of proteins and dephosphorylation of mononucleotides, phospholip-
ids and proteins. Furthermore, autolysis involves chromatin compac-
tion, nuclear fragmentation (involving RNA and DNA nucleases) and
cytoplasmic condensation and fragmentation. Thus, in FTIR-based
biomechanistic studies, where researchers are interested in identify-
ing the metabolites formed as a result of the cell’s response to specific
stimuli, the effects of autolysis as a consequence of inappropriate cell
preparation may obscure these investigations.
In cell biology, a critical and fundamental step in any investiga-
tion is “fixation.” This is used to quench autolysis, minimize leach-
ing of biomolecular constituents, whilst at the same time using
optimized dehydration protocols to bypass surface tension distor-
tions and preserve the structural and functional chemistry of bio-
molecules for analysis. The common methods of cell preservation
involve chemical fixation or flash-freezing for subsequent freeze-
drying. Flash-freezing is appropriate for cells grown on substrates,
which have good thermal contact with the freezing liquid medium
and substrates that can withstand the low temperatures involved
during this process. A common culture substrate for reflectance
mode measurements are low-e microscope slides, for example, the
MirrIR plate (Kevley Technologies). These slides are ~95 percent reflect-
ing in the mid-IR but ~80 percent transparent to visible light. This
makes them ideal for investigating biological cells and tissue, which
are best observed on the microscope slide using back-illumination.
They are also significantly cheaper than CaF or BaF plates. MirrIR
2 2
slides are relatively thick (2 mm) and have a large thermal mass.
Thus, the insulating effect of the MirrIR slide can slow down freez-
ing rates, resulting in intercellular ice crystal formation during
freezing. This can cause mechanical damage by rupturing cell mem-
branes and lead to the discharge of cytoplasmic material into the extra-
cellular matrix.