Page 111 - Vibrational Spectroscopic Imaging for Biomedical Applications

Sample Pr eparation of Cells and T issue 87

cells (human hepatocellular carcinoma) treated with increasing doses
of an environmental toxin. In this study, posttreated cells were
detached from culture substratum using trypsin, followed by two
washes in PBS then kept as a suspension at 4ºC and measured with
SR-FTIR within 24 hours. Although the cool temperature minimizes
the enzymatic effects of autolysis, without fixation there may be bio-
chemical differences between cells at the zero time point compared to
cells stored in PBS for 24 hours, particularly for glycogen stores, since
the cells were in a nutrient deficient environment. Nevertheless, it
has been shown that spectra from these cells showed spectroscopic
−1
−1
changes in the ratio of peak intensities (1082 cm /1236 cm ) that
could be correlated with increasing doses of toxin exposure. In a situ-
ation where the effect of time on cell biochemistry has not been
assessed, one must be careful when associating spectral changes to
the direct result of a condition administered to the cell. However, if
spectral discrimination is achieved following randomized sampling
of cells exposed to each of the different conditions, then this may
evaluate whether live cell spectra are significantly influenced by their
duration in nutrient deficient media.
More recently, specialised equipment for maintaining live T-1
cells (aneuploid cells from human kidney tissues) on gold-coated
slides for in situ SR-FTIR analysis has been investigated by Holman
58
et al. A mini-incubator system was used to sustain cell viability by
maintaining a humidified environment, so as to retain a thin layer of
growth medium around the cell during SR-FTIR measurements. The
mini-incubator was temperature controlled at 37°C via circulating
water from a water bath, and infrared transparent CaF windows on
2
the top cover were separately temperature controlled to avoid con-
densation. Using this incubator, the authors investigated any possible
cytoxic effects that may be elicited in the cell by exposure to the SR-IR
radiation. Using the Alcian blue exclusion assay, it was found that the
cells showed negative staining 24 hours after exposure to 20 minutes
of SR-IR radiation, which indicated that the cell membranes remained
intact. The effects of 20-minute SR-IR exposure on cell metabolism
was assessed using the MTT assay. This confirmed that both control
cells (not exposed to SR radiation) situated nearby to exposed cells and
exposed cells produced mitochondrial dehydrogenases, which is asso-
ciated with glycolysis and indicates negligible effects on this metabolic
pathway. Finally, colony-forming assays demonstrated that there was
no long-term damage as a result of SR-IR exposure.
Although these assays could not have been carried out in situ
within the mini-incubator, it is encouraging to find that the length
of time (20 minutes) that these cells were placed in the incubator
had no short-term or long-term effects. Furthermore, the research-
ers report that consecutive SR-FTIR spectra obtained at 10-minute
intervals for 30 minutes exhibited an unchanging IR spectrum to
within 0.005 A.U. across the entire mid-IR spectral range. This provides