Page 116 - Vibrational Spectroscopic Imaging for Biomedical Applications

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detachment when compared to a control monoculture. Raman spec-
tra obtained from different locations in the nanoparticle-doped cell
gave rise to very different spectral profiles, illustrating the biochemi-
cal heterogeneity of the cell (Fig. 3.14b).
As well as the application of Raman microspectroscopy to live
cell imaging, the technique has also been applied for the phenotypic
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typing of live cells. Krishna and colleagues collected Raman spectra
of two different cell lines and their respective drug resistant ana-
logues: Breast cancer cell line MCF7 and its subclone resistant to vera-
pamil (MCF7/VP) and promyelocytic leukemia HL60S cell line and
its multi-drug-resistant phenotypes (HL60/DOX: resistant to doxo-
rubicin; HL60/DNR: resistant to daunorubicin). PCA analyses of
these Raman spectra were able to generate score plots that showed
clustering and separation for each cell line and its drug-resistant
clone. The authors also carried out these experiments using FTIR and
found that classification, discrimination as well as reproducibility
was greater using this method. However, with the view of translating
this type of analysis to clinical application, it would be desirable for
the chosen method to incorporate minimal sample preparation for
high-throughput screening. For the Raman study, a cell pellet consist-
6
ing of 1.10 cells (washed in 0.9 percent NaCl) was used directly for
spectroscopic analysis, whereas for the FTIR experiments a time-limiting
step was required that consisted of drying a cell suspension under mild
vacuum onto a zinc selenide sample wheel.
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The sample preparation method used by Krishna et al. for the
Raman study requires fast data acquisition times, since live cell pel-
lets surrounded by a thin layer of aqueous buffer may undergo bio-
chemical changes over time. In their study, 25 spectra were collected
for each pellet, where one spectrum took 4.5 minutes to collect. Thus,
between the first and final spectrum there was a time lag of 1 hour
and 53 minutes. If biochemical changes did occur during this period,
then it may have contributed to the lower discriminatory power
achieved using Raman spectra in this study. Comparatively, the FTIR
spectra were obtained from dried cells, providing perhaps a back-
ground interference that is constant over all cells and so differences
due to MDR or drug sensitive phenotypes could be more readily
resolved. This provides further evidence that possible artifacts from
the drying process do not hamper spectroscopic differentiation
between cells of differing phenotypes (as mentioned in Sec. 3.3.2).

3.4 Summary
It is clearly evident that sample preparation is a key aspect of the
experimental design, where thorough dissection of the issues involved
in the preparation of cells or tissue for spectroscopic analysis is essen-
tial to yield reproducible and biochemically relevant results. The con-
tinuing developments in tissue preservation for optimum detection