Raman Spectroscopy: A Tool for Tissue Engineering - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 415

cell behaviour and whether the novel materials developed are having their de-
sired effect on cell behaviour (and subsequent formation of functional tissue)
are, however, complex questions to which Raman spectroscopy may offer some
answers.

18.1.2 Advantages of Raman Spectroscopy

Raman spectroscopy can offer a number of advantages over traditional cell
or tissue analysis techniques used in the field of TE (Table 18.1). Commonly
used analytical techniques in TE include the determination of a specific en-
zyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expres-
sion of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or
proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry)
relevant to cell behaviour and tissue formation. These techniques require inva-
sive processing steps (enzyme treatment, chemical fixation and/or the use of
colorimetric or fluorescent labels) which consequently render these techniques
unsuitable for studying live cell culture systems in vitro. Raman spectroscopy
can, however, be performed directly on cells/tissue constructs without labels,
contrast agents or other sample preparation techniques.
The Raman spectra of a biological cell (Fig. 18.2 [3]) or tissue construct
contain a complex array of peaks which provide molecular-level information
about all the cellular and extracellular biopolymers (nucleic acids, proteins,
lipids and carbohydrates). Thus the Raman spectrum of a cellular compo-
nent, single cell or tissue construct, provides a unique biochemical spectral
“fingerprint”, which provides a “snapshot” of all the biomolecular compo-
nents of the spectral sample area. Raman imaging also provides informa-
tion on the spatial distribution of one or more chemical species within a
heterogeneous sample. Raman spectroscopy of a cell/TE construct thereby
provides a more detailed “global” information bundle than that obtained
by traditional cell biological techniques, whereby the expression of individ-
ual genes or proteins is studied. However, the very information-rich nature
of this biochemical technique leads to extremely complex spectra, which re-
quires careful interpretation and analysis to extract valid and quantitative
data [4].
The effect of laser irradiation on cells during Raman spectral analysis
is contested and is certainly dependent upon laser wavelength, power and
the use of enhancement molecules (e.g. SERS particles) [5]. It is gener-
ally accepted, however, that NIR excitation (

700-1064 mm) is preferred for
non-invasive analysis of biological samples, as visible wavelength excitation
of live samples is plagued by fluorescence interference, and UV excitation-
associated biological damage by photo-degradation.
Fluorescent labelling of protein/RNA or DNA combined with optical or
confocal microscopy imaging is a powerful widely used imaging technique
that can (like Raman spectroscopy) also be applied to live cells or tissues.
This technique, however, is limited by the number of fluorescent molecular

∼