Biomedical Engineering Reference
In-Depth Information
FTIR has also been used for cell [7], tissue [8, 9] and biomaterial character-
isation [10]. Since FTIR spectroscopy and imaging is also derived from the
intrinsic molecular vibrational energy levels of a sample, like Raman spec-
troscopy, no external markers, dyes or labels are required for sample charac-
terisation. However, the
m spatial resolution provided by broadband
IR beams used in FTIR spectroscopy is significantly inferior to that offered
by diffraction-limited confocal Raman microspectroscopy (
∼
10-20
μ
m). Further-
more, while the weak Raman scattering of water enables spectral analysis
of hydrated biological samples (thereby allowing live, in situ measurements),
the strong absorbance of water throughout the infrared region in FTIR spec-
troscopy requires either fixation and/or dehydration of samples, or omission of
distorted spectral regions, both of which have the potential to induce artefacts
(Fig. 18.2). Transmission FTIR measurements also require very thin samples,
which necessitates invasive processing (e.g. sectioning) of biological samples.
Alternatively, attenuated total reflectance (ATR) FTIR can be used to anal-
yse sample surface properties. This technique, however, requires contact with
an ATR crystal, which can physically damage delicate biological samples.
∼
1
μ
18.1.3 Raman Spectroscopy Applied to Tissue Engineering
The recent boom in bioanalytical applications of Raman spectroscopy has un-
surprisingly coincided with technological developments in laser sources, key
spectrometer components (e.g. holographic notch filters), charged coupled de-
vice detectors and optimisation of collection geometries. These technological
advances have somewhat overcome the strong fluorescent interferences from
biological samples and the inherently weak magnitude of the Raman effect.
Furthermore, the development of enhanced and non-linear Raman techniques,
such as resonance Raman scattering (RRS), surface-enhanced Raman scatter-
ing (SERS), surface-enhanced resonance Raman spectroscopy (SERRS), co-
herent anti-Stokes Raman scattering (CARS) and FT-Raman spectroscopy,
offers exciting new opportunities in molecular imaging of biological samples
by reducing fluorescence and/or increasing the Raman scattering, resolution
and sensitivity [6, 11, 12].
In TE, non-resonant spontaneous Raman spectroscopy has, however, some
advantages over the enhancement techniques listed above, including the use
of relatively inexpensive excitation lasers, instrument compactness, robust-
ness, mobility, lower technical complexity and broadband ability (as all vi-
brational modes are excited simultaneously). Furthermore, enhanced RRS
and SERS techniques require culture on a metal substrate or cellular up-
take/membrane attachment of enhancement molecules, which may influence
cell behaviour thereby inhibiting the non-invasive nature of spontaneous Ra-
man spectroscopy. Moreover, while CARS produces a directed laser-like sig-
nal beam that is many times stronger than spontaneous Raman scattering
and avoids fluorescence interference (which occurs exclusively with Stokes sig-
nal), a technically complex spectrometer is required and multiplexed chemical
spectral resolution is limited. FT-Raman systems with noisy InGaAs detectors,
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