Raman Spectroscopy: A Tool for Tissue Engineering - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 427

18.5 Challenges and Limitations

18.5.1 Sample Imaging, Spectral Acquisition Time and Laser
Damage

Many 3D scaffolds used for TE are light impenetrable, thereby making the se-
lection of (suitable) sample sites, laser focussing and the interpretation of scaf-
fold features with spectral signatures somewhat problematic. SEM combined
with Raman offers a high-resolution solution to this problem but requires
fixed or frozen samples. In terms of Raman spectroscopic analysis of live cell
culture systems, a trade-off between spectral acquisition time and spectral
resolution occurs. This is a particularly pertinent issue for live cell or tissue
spectral analysis, where without an adequate sterile environmental chamber
(37 ◦ C, 5% CO 2 ) cells will respond to the environmental changes which will
cause phenotypic changes (e.g. cold shock) and artificial spectral signatures.
Furthermore, depending upon laser wavelength and power, increasing spectral
acquisition time will eventually cause laser damage to the biological system.

18.5.2 Scaffold Substrate

A further issue for Raman spectroscopy in TE is substrate/scaffold spec-
tral interference, whereby the Raman signal from the scaffold (e.g. polymer)
overwhelms the much weaker Raman scattering from cellular and/or ECM
biopolymers. For this reason most Raman cell studies use substrates (e.g.
CaF 2 ,MgF 2 and fused silica) which have low Raman signals in the biochem-
ical spectral regions of interest. Where multiple cell and protein layers exist,
substrate interference is somewhat diminished depending upon laser spot size,
substrate, depth of biopolymer layer and Raman set-up. Certainly a reduced
laser spot size, a confocal Raman spectroscopy set-up and spatially offset
Raman spectroscopy (SORS), allow both depth profiling and reduced sub-
strate spectral interference [44]. Reduced laser spot sizes will, however, invari-
ably increase spectral acquisition time, which may again cause issues for live
construct/cell analysis.

18.5.3 Total Tissue Construct or Surface Characterisation

Raman spectroscopic imaging of tissue-engineered constructs (using standard
instrument set-ups) is largely restricted to surface or near-surface biochemical
imaging. For example, for live cell or engineered tissue analysis our group (and
others) use a spontaneous Raman system with a 785 nm, 100 mW excitation
laser whereby the depth of imaging is approximately 40 m. While Raman

spectroscopic surface characterisation remains a valuable technique in the field

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