Phytochemistry 72 (2011) 1889–1895

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Distribution of lignin and its coniferyl alcohol and coniferyl aldehyde groups
in Picea abies and Pinus sylvestris as observed by Raman imaging
Tuomas Hänninen, Eero Kontturi ⇑, Tapani Vuorinen
Department of Forest Products Technology, School of Science and Technology, Aalto University, P.O. Box 16300, 00076 Aalto, Finland

a r t i c l e i n f o a b s t r a c t

Article history: Wood cell wall consists of several structural components, such as cellulose, hemicelluloses and lignin,
Received 11 February 2011 whose concentrations vary throughout the cell wall. It is a composite where semicrystalline cellulose
Received in revised form 27 April 2011 fibrils, acting as reinforcement, are bound together by amorphous hemicelluloses and lignin matrix.
Available online 31 May 2011
Understanding the distribution of these components and their functions within the cell wall can provide
useful information on the biosynthesis of trees.
Keywords: Raman imaging enables us to study chemistry of cell wall without altering the structure by staining the
Chemical distribution
sample or fractionating it. Raman imaging has been used to analyze distributions of lignin and cellulose,
Coniferyl alcohol
Coniferyl aldehyde
as well as the functional groups of lignin in wood.
Picea abies In our study, we observed the distribution of cellulose and lignin, as well as the amount of coniferyl
Pinus sylvestris alcohol and aldehyde groups compared to the total amount of lignin in pine (Pinus sylvestris) and spruce
Raman imaging (Picea abies) wood samples. No significant differences could be seen in lignin and cellulose distribution
between these samples, while clear distinction was observed in the distribution of coniferyl alcohols
and coniferyl aldehyde in them. These results could provide valuable insight on how two similar wood
species control biosynthesis of lignin differently during the differentiation of cell wall.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction since the activity of some coniferyl aldehyde dehydrogenase

Lignin in the wood cell wall has several functions. While being a
binding component between individual cells, it also controls the
water content inside the cell wall enabling transport of water
and providing protection against pathogens as well as strength to
load-bearing structures (Iiyama et al., 1994; Boerjan et al., 2003).
Lignin is commonly defined as a complex hydrophobic network
of phenylpropanoid units derived from the oxidative polymeriza-
tion of one or more of the three types of hydroxycinnamyl alcohol
precursors (p-hydroxyphenyl, guaiacyl and syringyl units). Such
definition, however, has been shown to be insufficient, for it is
likely that no plant lignin derives solely from the three precursors
(Sederoff et al., 1999).
Hypothetically, the structure of lignin is determined by the rel-
ative abundance of the precursors in the lignifying zone. This is
controlled by genetic and environmental factors (Boerjan et al.,
2003). One of the functional groups that has received relatively lit-
tle attention from a structural perspective is coniferyl aldehyde
(lignin-CAld), the reduction of which is considered to be the final
step in the biosynthesis of coniferyl alcohols (lignin-CAlc) in lignin
(Boerjan et al., 2003). Lignin-CAld groups prevail in native lignin
⇑ Corresponding author. Tel.: +358 9470 24250; fax: +358 9470 24259.
E-mail address: eero.kontturi@aalto.fi (E. Kontturi).
(CAD) enzymes is reduced during lignin biosynthesis (Boerjan
et al., 2003). Coniferyl aldehydes have been shown to inhibit cell
wall degradation by enzymes (Grabber, 2005), and they have been
proposed to be a plant’s response to a wound (Kim et al., 2003) or
biotic and abiotic stress (Barakat et al., 2009).
To better understand the structure and function of lignin, a look
at the broader composition of the wood cell wall is useful. Lignin is
associated with other cell wall polymers, namely cellulose, hemi-
cellulose and pectin. The latter two polymers combine with lignin
to form the individual cell binding matrix known as the middle
lamella whereas cellulose is the most important load-bearing com-
ponent of the cell wall. Cellulose in plants is found as semi-crystal-
line aggregates, microfibrils, whose orientation along the fiber axis
varies depending on their location in the cell wall.
During the cell formation, the biosynthesis forms lamellar cell
wall layers which differ from each other in contents of structural
components and their alignment. The division of a cell wall into
two major layers is fairly established: the secondary cell wall re-
sides next to the hollow lumen, which is further divided into three
layers S3, S2 and S1, and outside the primary cell wall layer forms a
thin cover for the cell. The structure of wood cell wall is schemat-
ically depicted in Fig. 1 which clearly shows the division in differ-
ent cell wall layers and changes in cellulose microfibril orientation.
The distribution of lignin and other structural components of
the wood cell wall has commonly been studied by using optical

0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.05.005
1890 T. Hänninen et al. / Phytochemistry 72 (2011) 1889–1895

wall level, which has not been shown earlier. These two species are
the most common and industrially most important softwood spe-
cies in the Nordic countries. The direct visualization of various
components and functional groups within the cell wall is bound
to yield fresh knowledge on their role in plants. Furthermore, by
understanding the variations in chemical distributions of cell walls,
industrial processes can be tailored more specifically for certain
species. For example, lignin-CAld contents in wood have been
shown to affect the result of chemical pulping (MacKay et al. 1999).

2. Results and discussion

2.1. Lignin/cellulose distribution

A Raman spectrum consists of bands which are caused by an

inelastic scattering from the chemically bonded structures. Cellu-
lose, hemicelluloses, pectins and lignin all contribute to the Raman
spectrum collected from wood. Raman spectra from the secondary
cell wall of spruce and pine are presented in Fig. 2A and B to illus-
trate the complexity of wood spectrum. Cellulose and lignin pro-
vide the most prominent Raman bands, while hemicelluloses and
pectins have remained undetected due to their low content, broad
Raman bands and overlapping with stronger bands of other com-
Fig. 1. Schematic representation of the structure of wood cells. Individual cells are ponents (Agarwal and Ralph, 1997). Spectra of pure model compo-
separated by middle lamella. The cell wall is divided into two layers, primary wall nents have been measured in several studies in order to detect
(P) next to middle lamella (ML) and secondary wall which is further divided in S1, their contributions in the Raman spectra of lignocellulosic sub-
S2 and S3 layers. A hollow lumen is surrounded by the cell wall layers. stances (Agarwal and Ralph, 1997, 2008; Wiley and Atalla, 1987;
Saariaho et al., 2003a,b; Nuopponen et al., 2004a,b).
Polarized Raman spectra of polymers are sensitive to orienta-
(Ritter, 1925; Wardrop, 1965; Scurfield, 1967; Côté et al., 1968;
tion (Koenig, 1999), which must be considered in the interpreta-
Peng and Westermark, 1997) and electron microscopy (Fernando
tion of their spectra. Especially the Raman spectrum of cellulose
and Daniel, 2008; Terashima et al., 2009) or preparative fraction-
has been shown to change in different orientations (Atalla et al.,
ation of cell wall sublayers (Marton and Adler, 1961; Whiting
1980), while the spectrum of lignin shows only small changes
and Goring, 1983; Sorvari et al., 1986; Sundberg et al., 2002). When
(Atalla and Agarwal, 1985). The sensitivity toward orientation in
a wood sample is fractionated or modified to be suitable for micro-
Raman bands of cellulose has also been applied to determine the
scopic analysis, mechanical or chemical treatment is applied to the
microfibril angle in different cell wall layers, as shown in a recent
sample. This can cause changes in the cell structure and modify cell
study by Gierlinger et al. (2010). To avoid misinterpretation of the
wall components introducing artifacts to the sample. By using
results due to orientation, our measurements have been conducted
spectroscopic methods, such as infrared (IR) and Raman spectros-
so that the measured area contains cell walls that are along and
copies, measurements can be performed on the samples without
perpendicular to the polarization of the excitation laser, in the
any preparatory steps that could alter them. Measurements on
images vertical and horizontal cell walls, respectively.
wood in its native state are possible with Raman spectroscopy, be-
When analyzing images consisting of the intensity of certain
cause the Raman response of water is very weak due to its low
bands only, it is necessary to understand that the presented val-
polarizability, i.e., water does not dominate over the Raman spec-
trum and therefore disturb the measurement. In contrast, an IR
spectrum is significantly influenced by the presence of water in
the sample since IR absorption is pronounced for polar molecules.
Limits of resolution in IR become more undesirable when consid-
ering spectroscopic imaging. In spectroscopic imaging, for example
Raman imaging, spectra are collected at regular, spatial intervals
from the selected area on the sample. From the collected spectra,
images are constructed according to the heights or areas of the se-
lected bands. Such images illustrate changes in chemical structures
or distributions of different components in the sample. Raman imag-
ing has been shown to provide informative results when interpret-
ing the cell wall ultrastructure of wood (Agarwal, 2006; Gierlinger
and Schwanninger, 2006; Schmidt et al., 2009; Gierlinger et al.,
2010) and other plant (Gierlinger and Schwanninger, 2007) samples.
Earlier very few studies on the distribution of lignin-CAld (and
lignin-CAlc) groups in wood cell walls (Wardrop and Davies,
1959; Peng and Westermark, 1997; Pomar et al., 2002) have been
conducted. In the present study, we used Raman microscopy to
determine the distribution of lignin and, more importantly, the dis- Fig. 2. Raman spectra from S2 cell wall (A) and cell corner middle lamella (B) of
pine. Spectra from middle lamella of NaBH4 treated pine (C) and spruce (D) samples.
tribution of lignin-CAld and lignin-CAlc groups in the cell wall of Spectra have not been normalized. Spectra shown in figure are averages of several
two softwood species, Scots pine (Pinus sylvestris) and Norway spectra collected from data used for images. Spectra of single measurements are
spruce (Picea abies). Clear differences could be distinguished in cell presented in Supplementary material Fig. S2 online.
 T. Hänninen et al. / Phytochemistry 72 (2011) 1889–1895 1891

ues do not necessarily correspond to the actual amounts of that
compound in the sample. Many factors, such as an uneven
surface, cracks in the sample and changes in the intensity of exci-
tation laser, can affect spectra, thus introducing artifacts to
images. Images constructed solely according to the intensity of
Raman bands are presented in the Supplementary material
Fig. S1 online.
The images constructed according to 1600 cm 1 band repre-
senting lignin (Supplementary material Fig. S1A and D), indicate
rather even distributions in the secondary cell wall while the pri-
mary cell wall and the middle lamella contain higher concentra-
tions of lignin in all of the wood samples. Images of cellulose
distribution, constructed according to 1095 cm 1 band (Supple-
mentary material Fig. S1B and E), show an even distribution in
the secondary cell wall while the cellulose content in the middle
lamella is significantly lower. The region of S1 and primary cell
wall exhibit changes in the intensity in vertical and horizontal cell
walls which is an artifact caused by the changes in the orientation
of cellulose with respect to excitation light polarization. This
phenomenon has been elaborated earlier by Wiley and Atalla
(1987) and utilized more recently in Raman imaging by Agarwal
et al. (2007) and Gierlinger et al. (2010). The impact of microfibril
orientation on the Raman spectra is fundamentally important. In
this work, however, we have focused on the distribution of various
chemical components and functional groups in the cell wall, partic-
ularly that of lignin-CAld and lignin-CAlc.
By using ratios between two Raman bands, artifacts caused by
the uneven surface and changes in the laser intensity can be over-
come. Raman band intensity ratios of components are not altered
due to changes in the measurement parameters. Images con-
structed according to the ratio between lignin and cellulose Raman
bands, depicted in Fig. 3A and D, exhibit very similar features to the
images constructed by using single bands. Distribution is even in
the secondary cell wall while in the middle lamella the lignin/cel-
lulose-ratio is much higher. Artifacts caused by cellulose microfi-
bril orientation, which manifest themselves as darker (horizontal
cell walls) and lighter (vertical cell walls) areas in the region of pri-
mary and S1 cell wall layers, can also be seen in these images.

Fig. 3. Raman images of transversally sectioned pine and spruce. Raman images of pine (A–C) and spruce (D–F) illustrate lignin/cellulose, lignin-CAA/lignin and lignin-CAld/
lignin-ratios within the cell walls. Confocal micrographs of samples are presented in Supplemental Fig. 3 online.
1892 T. Hänninen et al. / Phytochemistry 72 (2011) 1889–1895
Overall, the results obtained resemble the lignin and cellulose
distributions obtained recently with Raman imaging from a variety
of wood species (Agarwal, 2006; Gierlinger and Schwanninger,
2006; Schmidt et al., 2009; Gierlinger et al., 2010). A comparison
between pine and spruce, as well as with the previously published
works, points towards the direction that the lignin/cellulose distri-
bution within the cell wall could be independent of wood species.
Indeed, species as versatile as conifers (this study; Agarwal, 2006;
Gierlinger et al., 2010) and deciduous trees (Gierlinger and
Schwanninger, 2006; Schmidt et al., 2009) appear to possess rela-
tively similar lignin/cellulose distributions. Changes in distribution
seem to emerge only when the structure of the cell is altered, for
example, in compression (Gierlinger et al., 2010) or in tension
wood (Yoshida et al., 2002; Gierlinger and Schwanninger, 2006).
This is in line with previous results obtained by a variety of analyt-
ical techniques (Wardrop, 1965; Côté et al., 1968; Fergus et al.,
1969). However, Raman imaging is inherently a somewhat robust
technique and the diffraction limit prevents the observation of re-
gions smaller than ca. 300 nm. In fact, slight anomalies of lignin
distribution throughout the cell wall have been observed in trans-
mission electron microscopy (TEM) (Côté et al., 1968; Fernando
and Daniel, 2008). For example, a layer enriched in lignin was
found in S1 layer in spruce (Fernando and Daniel, 2008).
2.2. Coniferyl alcohol and aldehyde group distribution
The distributions of coniferyl alcohol and aldehyde (joint abbre-
viation lignin-CAA for both structures) were scrutinized in this
work because of their scarce appearances in the past literature
and their relatively effortless distinction in the Raman spectrum
of wood. As reported by Agarwal and Ralph (2008), only aromatic
ring-conjugated ethylenic and carbonyl structures contribute to
Raman bands at ca. 1660 cm 1. The lignin-CAA structures are diffi-
cult to resolve from each other due to the overlapping of lignin-
CAlc and lignin-CAld bands.
Constructing Raman images merely according to the intensities
of the lignin-CAA band at 1660 cm 1 yields very little information
on the actual distribution of lignin-CAA within the cell wall. In fact,
these images resemble those that are constructed according to the
band at 1600 cm 1 representing all aromatic lignin structures (see
Supplementary material Fig. S1C and F). Extractives containing
aromatic structures could also contribute to the wavenumber area
around 1600 cm 1 (Nuopponen et al. 2004a,b). They have been,
however, removed from the sample upon the solvent exchange
during the sample preparation.
In order to visualize distributions of different functional groups
of lignin, we chose to utilize the relative band intensities with re-
spect to the band at 1600 cm 1. Images illustrating lignin-CAA
group/lignin and lignin-CAld/lignin-ratios in untreated wood and
NaBH4 treated samples are shown in Fig. 3B, C, E, and F, and
Fig. 5B, C, E, and F, respectively.
With this approach we exploited the feasibility to reduce the
lignin-CAld into lignin-CAlc and thereby attempted to gain infor-
mation on the changes of their ratios within the cell wall by per-
forming Raman imaging before and after the NaBH4 reduction.
The reaction scheme of NaBH4 reduction is presented in Fig. 4.
Although both lignin-CAlc and lignin-CAld groups contribute at
1660 cm 1, their relative intensities differ, which enables us to
see the changes in the samples after the reduction (Agarwal and
Ralph, 2008). This is also visible in Fig. 2.
The images constructed according to lignin-CAA/lignin-ratio
show clear differences between pine and spruce (Fig. 3B and E,
respectively). In spruce, the secondary cell wall lignin contains a
higher amount of lignin-CAA groups than the lignin in the middle
lamella and in the primary cell wall area. In pine, however, the pri-
mary cell wall area lignin seems to contain a lower amount of
Fig. 4. Reaction scheme for the reduction of coniferyl alcohol into conifer aldehyde
by NaBH4.
lignin-CAA groups than the lignin in the middle lamella. The sec-
ondary cell wall of pine seemed to contain a higher amount of lig-
nin-CAA than the primary cell wall area and roughly the same
amount as the middle lamella.
After the reduction of lignin-CAld groups to lignin-CAlc with
NaBH4, the differences between species disappear and the distribu-
tions of lignin-CAA groups in both species (Fig. 5B and E) resemble
the one from the untreated spruce (see Fig. 3E). From this we can
deduce that primary cell wall and the middle lamella of pine con-
sist of two different kinds of lignin with varying amount of lignin-
CAld groups, while the lignin-CAld distribution in this region is
more uniform in spruce. Indeed, these results suggest that the lig-
nin in the region around the primary cell wall of pine contains a
higher amount of lignin-CAld groups than lignin located in the
other cell wall layers. This hypothesis is supported by the disap-
pearance of the residual layer with low lignin-CAA/lignin band ra-
tio after NaBH4 treatment of pine (Fig. 3B vs. Fig. 5B).
Agarwal and Ralph (2008) determined a characteristic Raman
band for only coniferyl aldehyde structures at 1623 cm 1, which
was used to construct images illustrating lignin-CAld/lignin distri-
butions (Fig. 3C and F, Fig. 5C and F). In both pine and spruce sam-
ples, images illustrating lignin-CAld/lignin-ratio (Fig. 3C and F)
show an increased amount of lignin-CAld groups roughly in the re-
gion of the middle lamella and the primary cell wall. However, the
resolution is very poor due to a low signal-to-noise ratio which
renders more detailed conclusions concerning lignin-CAld group
distribution impossible.
After the NaBH4 treatment, the lignin-CAld/lignin-ratio is even
throughout the cell wall for both of the sample wood species
(Fig. 5C and F). This indicates that the NaBH4 treatment has indeed
reduced all of the lignin-CAld groups in the samples, since the sep-
arate band at 1623 cm 1 disappears. In other words, the intensity
contribution at 1623 cm 1 arises from the decaying profile of the
band peaking at 1600 cm 1, which causes the ratio between the
bands to be nearly the same throughout the cell wall. The absence
of 1623 cm 1 band is also apparent in the spectra of Fig. 2C and D.
Furthermore, a new, prominent Raman band arises after the
reduction at around 1635 cm 1 in pine samples only (Fig. 2C). Such
a Raman band has not been reported earlier for lignin, but it has
been found to occur in stilbene samples (Holmgren et al., 1999;
Agarwal and Landucci, 2004). It has also been calculated that feru-
lic acids have a Raman band at that wavenumber area (Sebastian
et al., 2009). Model compound studies would be required to deter-
mine which chemical structure is presented by the 1635 cm 1
band. However, this is beyond the scope of this study.
Earlier studies of phloroglucinol-HCl stained spruce cell wall by
UV–Vis microscopy have shown higher lignin-CAA group contents
in the primary wall and S1 layer and, additionally, lignin-CAlc
enrichment in the S3 layer (Peng and Westermark, 1997). Another
study by Agarwal (2006) from a related species (Picea mariana) car-
ried out with Raman imaging did not reveal any distinctions in the
lignin-CAA contents of different cell wall layers. These authors,
 T. Hänninen et al. / Phytochemistry 72 (2011) 1889–1895
however, did not apply the relative amount of lignin-CAA groups
with respect to the total amount of lignin. The relative lignin-
CAA content was applied successfully by Schmidt et al. (2009)
but their study focused on the transgenic deciduous species.
The reason why the distribution of lignin-CAlds in particular
differs between spruce and pine is difficult to address. Although
lignin-CAlds have been studied for a long time (Hibbert, 1942;
Adler et al., 1948), their function in lignin is still under discussion.
Increased amounts of coniferyl aldehydes have been found in CAD
deficient trees. CAD deficiency alters the color of the tree slightly
(Ralph et al., 1997), but no changes can be seen in the mechanical
properties (Saralde et al., 2008). It has also been speculated that an
increased amount of aldehyde groups in lignin could be a result of
tree’s response to a wound (Kim et al., 2003).
Lignin-CAld groups may also bear implications for the utiliza-
tion of wood. Because of their nature as terminal groups, lignin
with high content of aldehydes has been reported to have a de-
creased molar mass (Kim et al., 2003), which affects the mechani-
cal properties of lignin. The decreased molar mass increases the
Fig. 5. Raman images of transversally sectioned NaBH4 reduced pine and spruce. Raman images of pine (A–C) and spruce (D–F) illustrate lignin/cellulose, lignin-CAA/lignin
and lignin-CAld/lignin-ratios within the cell walls.
1893
mobility of lignin, which in turn lowers the glass transition tem-
perature (Olsson and Salmén, 1997). This could affect the behavior
of wood in processes operating at high temperatures, such as ther-
momechanical pulping. The amount of coniferyl aldehydes has also
been shown to affect pulping (MacKay et al., 1999).
To summarize, the present work indicates, with the aid of a
non-destructive spectroscopic imaging method and several litera-
ture accounts, that lignin/cellulose-ratio is largely similar in sev-
eral common wood species, whereas the distribution of lignin
structures can already differ between two relatively similar north-
ern conifers. This finding bears implications on the functions of dif-
ferent components and structures within the cell wall matrix and it
may help interpret their biosynthetic origin.
3. Experimental
The wood samples were embedded in epoxy resin (Agar Low
Viscosity Resin, Agar scientific, Essex, England), subsequent to
1894 T. Hänninen et al. / Phytochemistry 72 (2011) 1889–1895
Table 1
Raman band regions of compounds used for construction of images. Bands are chosen
according to several publications (Agarwal and Ralph, 1997, 2008; Gierlinger and
Schwanninger, 2006).
Compound Wavenumber region Raman mode
(cm 1)
Lignin 1583–1620 Aromatic ring mode
Cellulose 1090–1100 C–O stretch
Epoxy resin 1726–1754
Coniferaldehyde and alcohol 1649–1677 C@C stretch
(lignin-CAA)
Coniferaldehyde 1623–1633 C@C stretch
ethanol-acetone-resin solvent exchange, and sectioned with a dia-
mond knife using an ultramicrotome. Solvent exchange was per-
formed by keeping the samples in 1:0, 3:1, 1:1, 1:3 and 0:1
ethanol:acetone mixtures for about 10 min, consecutively. Subse-
quently samples were kept in 3:1, 1:1, 1:3, 0:1 acetone:resin mix-
tures in vacuum for about 1 h and the agitated gently over 6 h. No
hardener was used in the resin during solvent exchange sequence
before the final step. In the final step, the samples were added in re-
sin with hardener, kept in vacuum for about 1 h and cured in 60 °C
overnight. Sections, whose thickness varied from 5 to 9 lm, were
cut on water and then they were moved on the glass plate for Raman
measurements. For the samples whose lignin-CAld groups were
transformed to lignin-CAlc groups, matchstick size pieces of wood
were treated with 0.5 M NaBH4 for 4 h at room temperature. After
the reaction, the excess NaBH4 was removed by setting the pH of
the solution to 6 by using HCl and subsequent rinsing. This method
was adapted from Agarwal and Ralph (2008) by changing reaction
times to be more suitable for solid wood samples.
Only latewood fibers were measured, because the dimensions
of earlywood cell walls are too small to yield valuable information
regarding the diversity of the cell wall structures. The samples con-
sisted of both juvenile and mature wood; however, in this study
they were not considered separate sample groups.
The samples were analyzed with an alpha300 R Confocal Raman
microscope (Witec GmbH, Germany, www.witec.de) at ambient
conditions. The Raman spectra were obtained by using a frequency
doubled Nd:YAG laser (532.35 nm, 10 mW) and a Nikon 100x
(NA = 0.95) air objective. The Raman system was equipped with a
DU970 N-BV EMCCD camera behind a 600 lines/mm grating. The
excitation laser was polarized horizontally. For each single spec-
trum in the Raman images, an integration time of 0.3 s was used.
The size of one pixel in image is 0.1 lm. The baseline of the spectra
were corrected with WiTec Project 1.94 (WiTec GmbH, Germany,
www.witec.de) by using the fifth order equation in the wavenum-
ber area from 600 cm 1 to 2000 cm 1, where the most prominent
Raman bands are located. Wavenumber areas used for the baseline
correction are presented in the Supplementary material Fig. S3. A
constant background was not subtracted from spectra due to the
varying baseline of the individual measurements.
The selected Raman wavenumber regions for area-intensity cal-
culations and their corresponding vibrational modes are presented
in Table 1. Raman images were constructed according to the inte-
grated area at the wavenumber region of characteristic bands. Ra-
man spectra for the images were collected at 100 nm intervals.
Such a high resolution was applied because some details appeared
only in the images with a higher resolution than the diffraction
limitation would allow.
Acknowledgements
This work was part of projects supported by Nordic Energy
zResearch (NER) and Multidisciplinary Institute of Digitalisation
and Energy (MIDE, http://mide.tkk.fi).
We also thank Oy Keskuslaboratorio-Centrallaboratorium Ab
(KCL) for providing us the samples and Ms. Rita Henriksen for
her help on illustration.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2011.05.005.
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