Applications of Raman and Surface-Enhanced Raman Scattering to the Analysis of Eukaryotic Samples - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 81

Hybridization has retained oaks a model taxon for species concepts that rely
on ecological criteria rather than on reproductive isolation [65]. The high sim-
ilarity in chemical constitution of different oak pollens, which is verified by a
very similar biochemical fingerprint in our Raman experiments, can be inter-
preted as part of the very weak barriers to being pollinated by another (oak)
species.
Raman spectra of pollen are fingerprints of their particular biochemical
composition. Therefore, they reflect part of the phenotype of a plant. To
summarize the discussion of the spectral classification result in the context
of phylogenetic groups investigated here, the similarities and dissimilarities
observed in the Raman spectra of the pollen are in accordance with concepts of
systematics and speciation. These are the processes of reproductive or mating
isolation (which is sensible, considering that pollen are critical parts of mating
systems), but also, as was observed here for the case of the Maple genus,
ecological and geographical criteria. It remains to be investigated whether the
latter separation represents environmental or evolutionary effects.

4.2.4 Influence of Resonant Raman Signals
from Carotenoid Molecules

From the cluster analyses we learn that the biochemical origins of the species-
specific fingerprints lie in all classes of molecules contained in the pollen grains.
However, the content in certain molecular constituents can interfere with the
meaningful classification of a sample. Initially, we noticed very intense contri-
butions from carotenoids in the Raman spectra of pollen from some species.
They led to a classification of the spectra according to carotenoid content in
multivariate analyses (Fig. 4.4). Carotenoids have been reported to be com-
monly involved in pollen pigmentation, mainly contained in the exine layer,
although early hypotheses that carotenoids and carotenoid esters polymerize
to form the sporopollenin polymer itself [66] have proven false [1]. However,
the outer pollen wall (exine) is an accumulation site for various pigments, and
in a recent report, CARS imaging of an individual cherry pollen grain was
based entirely on the ν 3 Raman modes of carotene [67].

It should be noted that our excitation wavelength of 785 nm is far away
from the absorption maxima of typical carotenoid molecules [68, 69]. We con-
clude that the intense spectral contributions of the carotenoids are caused by
both a relatively high carotenoid content in the pollen of particular species,
as well as pre-resonant enhancement, yielding carotenoid intensities that are
about one order of magnitude above those of the other bands, e.g. the rela-
tively strong CH 2 deformation mode at 1445 cm − 1 (Fig. 4.5, traces a, c and e).
The observed pre-resonant Raman signals of carotenoids can be orders of mag-
nitude stronger than the normal Raman signals of all other molecules in the
pollen.

ν 1 and