Biomedical Engineering Reference
In-Depth Information
biopolymer of largely unknown composition and structure, the sporopollenin
of their outer layer, to flavonoid and carotenoid pigments, lignin, pectin and
the constituents of the cells in the pollen interior, namely proteins, lipids, car-
bohydrates and nucleic acids [1, 2, 4, 44]. Many phenomena related to pollen
are not well understood yet, among them the composition of the aforemen-
tioned sporopollenin capsules and the local distribution of molecules. To date,
approaches to pollen chemical characterization involve purification, the latter
implying possible modification or extraction of selected pollen constituents
(allergens, carotenoids and lipids) [45, 46].
Different from identification of other biological entities, e.g. in bacterial
typing, current pollen identification procedures do not rely on molecular pa-
rameters, but mainly on morphological information obtained through optical
or electron microscopies. Morphological information from light microscopy
and image analysis is also the basis of current allergy warning systems. In
the light of the urgent need for more ecient pollen identification and early
warning systems due to an increased prevalence of pollen allergies [47], a fast
identification method based on objective chemical information is sought.
The first Raman spectroscopic work on whole pollen was carried out in res-
onance with electronic transitions in molecules mainly from the pollen exine
layer by Manoharan et al. [48]. Non-resonant Raman spectroscopy has been
proposed for studies of pollen for several years, but was based on feasibility
studies on the few freeze-dried pollen species that are commercially avail-
able [49-51]. Some of these spectra in the literature were excited at very high
laser powers of several watts and discussed very high fluorescence backgrounds
[49, 50], even at NIR excitation [51]. The idea of using vibrational spectro-
scopic methods for discrimination between pollen from different plant species,
similar to classification approaches for microorganisms, cells and tissue types
is not new. However, it must have been hampered by methodological problems,
in particular the fluorescence background. We could show that fresh pollen
samples, which are of particular relevance to the development of actual on-line
pollen detection methods, exhibit only low levels of fluorescence and enable
acquisition of high-quality Raman spectra [52]. A study on IR spectra of dried
pollen from different plants (trees, shrubs and grasses) demonstrated separa-
tion of different species within the
Citrus
genus [53]. In this case, however,
sample preparation (preparation of KBr pellets) was relatively complicated.
The few other vibrational studies of pollen conducted so far investigated the
spectra of different species that were also members of different genera, dif-
ferent families and oftentimes even different orders of plants [48, 49, 51, 53].
In a study using surface-enhanced Raman scattering (SERS) on four species,
which concluded that pollen of the same two plant families have similar SERS
spectra [54], the pollen did not only belong to different plant families, but also
to different orders, classes and even divisions in the plant kingdom.
All these studies left the question open whether distinction between Raman
spectra down to species level can really be achieved (within the same genus).
To find an answer to this question, we have included in our sample set a
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