Anal Bioanal Chem (2003) 376 : 512517

DOI 10.1007/s00216-003-1905-2

O R I G I N A L PA P E R

G. Holzhter K. Narayanan T. Gerber

Structure of silica in Equisetum arvense

Received: 27 September 2002 / Revised: 3 March 2003 / Accepted: 11 March 2003 / Published online: 6 May 2003
Springer-Verlag 2003

Abstract Silicified regions in the stem and leaf of the

horsetail Equisetum arvense were studied by scanning and
transmission electron microscopy. The silica was present
as a thin layer on the outer surface with variation in the
size of this layer depending on the part investigated. There
was a dense arrangement of silica spheres with some density fluctuations. A loose arrangement of silica particles
with variation in their size was found beneath this dense
arrangement suggesting the agglomeration of silica. An
electron diffraction pattern showed the presence of amorphous silica, with the short range order being comparable
to that of silica from other chemical sources. The medium
range order shows the presence of silica with a high inner
surface. SAXS measurements correlate with the particle
size observed in TEM, and provide values for surface
fractals. A new method of plasma ashing to remove the
organics is also described.
Keywords Silica Horsetail Biomineralisation
Electron microscopy SAXS

Introduction
In nature, silica can be found in higher terrestrial plants
and a wide variety of organisms like diatoms, sponges and
molluscs [1, 2]. De Saussure (in Ref. [3]) first described
the silicification in plants. Silica distribution in various
grasses [4, 5, 6] has been widely studied and reported. The
shapes of phytoliths are characteristic of a given plant and
vary between different species [7, 8]. Usually silica is
found in parts of plants associated with cell wall in the
subterranean and aerial organs, those that infill the cell lumen and as extra cellular deposits. These silica deposits
can reproduce or mimic carbon-based cell structures in

G. Holzhter () K. Narayanan T. Gerber

Department of Physics, University of Rostock,
18051 Rostock, Germany
e-mail: holz@physik1.uni-rostock.de

three dimensions; such replacement structures are retained

long after the death of the plant and can be used to identify the precursor plant. Although silica may not be necessary for healthy growth of the plants, it has been found to
have some secondary effects. It has been speculated that
biomineralised silica may play a structural role or act as a
mineral barrier to both invasion of pathogens and translocation of water and salts.
No other plant has a higher concentration of silica than
the horsetail [9]. It is one of the oldest plants on earth and
what remains today from tree-sized fossils are the field
horsetails. They were used in historical times for scouring
pots and polishing pewter and were commonly called
scouring rushes. Horsetails have found extensive application in medicine as a source of silica, as it can amount
to 25% of the dry weight of the plant. Researchers [10, 11]
believe that the medicinal property of horsetail is due to
its high silica content. It has good antibacterial, antiseptic
and astringent properties. In many plants dissolved silica
appears to be taken into the plant as an inert component
and is deposited in places where water evaporates. In any
case, the structure of silica determines the property and usage. But the mechanism of silica accumulation in the
plants [12] still remains poorly studied although various
possible mechanisms have been proposed. Previous research on silica distribution in horsetails was mostly based
on scanning electron microscopy [13] and the importance
of structurefunction relation and cell wall composition in
horsetail [14] has been discussed. The associated macromolecules which might be the structure-directing agents
seem to be unknown. Biosilicification and the role of the
organic matrix in controlling the structure has been reviewed in detail by Perry et al. [15].
In previous studies, mostly ground horsetail samples
were taken for study which does not focus on the point of
interest. In the current study, we examine sections across
various significant silica structures using a number of different methods for structural investigation. A structure correlation to the investigating methods has been attempted.

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Fig. 1 (a) Cross section through the
stem of equisetum arvense; scale bar
200 m. (b) Silicon distribution in
Fig. 1a

Experimental
Materials and methods
Samples of shoots of Equisetum arvense were collected in autumn
and spring. Shoots were separated into stems and leaves. A part of
the sample was fixed with glutaraldehyde and subjected to critical
point drying so as to retain the cell structures without significant
deformation. A part of this fixed sample was further fixed with
OsO4 for contrasting between the organic part and silica and then
embedded in epoxy resin. Ultrathin sections of both longitudinal
and axial parts were made with a diamond knife on an ultramicrotome and placed on copper grids. These sections were viewed in a
LEO 912 TEM operated at 100 keV and electron diffraction was
made on the same instrument. TEM images and the diffraction pattern were recorded on image plates. Gold was used as the standard
for calibration. From the diffraction pattern of the samples and
gold standard, radial intensity scans were made. The scattering intensities were normalised and then Fourier transformed to difference atomic radial distribution functions [16, 17, 18]. Samples
were mounted on aluminium stubs and coated with gold for analysis by scanning electron microscopy. For elemental mapping, the
samples were taken as such without gold sputtering. In order to remove the organics for improved examination of the silica layer,
natural samples (without any fixation) were plasma ashed in an
oxygen atmosphere in a K1050 Plasma asher. The samples were
plasma ashed for 4.5 h with a power of 150 W. Plasma ashed and
natural samples were lightly ground with a pestle and mortar and
the powdered sample was subjected to SAXS studies. SAXS measurements were performed on a Kratky system using Ni-filtered
Cu K radiation (sealed tube, 2 kW X-ray generator); data were
collected and analysed by a previously described protocol [19].

Results and discussion

Localisation of silica
Elemental mapping of silicon was done on samples that
had been subjected to critical point drying to identify positions where silica was concentrated. Studies on several
parts of the stem and leaves gave impressive results. Figure 1a shows the SEM of cross section of the stem from
Equisetum arvense. Figure 1b shows the silicon distribution in the regions of Fig. 1a. In general, the relative positioning of silica was as a thin layer on the surface. And
this thin layer was prominent, irrespective of the part of

Fig. 2 Fully mineralised outermost cell wall in the cross section of

stem; scale bar 2 m

the plant examined. This can be because, once the silica is

concentrated and converted to colloidal forms, it cannot
enter the cell membranes and so remains concentrated as
such.
Ultra structure studies by TEM
TEM examination showed that the silica surface layer
varied in thickness depending on the part of the plant, the
leaf or the stem. To describe the silica layer more thoroughly, we divided the layer into three parts an outermost thin layer, a dense middle region and an inner region.
In general, the silica layer was found to vary in thickness between 3 m and 7 m in the stem and from about
200 nm to 1 m in the leaf (Figs. 2 and 3). The outermost
thin layer was found to consist of particles which resemble spheres without pores. The size of these spheres varied between 25 nm and 40 nm in diameter. In the middle
region, arrangement of the silica was found to be more
compact, but there were some density fluctuations in the
distribution.
When the middle layer is viewed under a higher magnification, new structures with some holes and channels in

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Fig. 5 The inner part of the mineralised layer consisting of 60 nm

spheres and 8 nm silica particles; scale bar 100 nm
Fig. 3 Fully mineralised surface region of a leaf cross section;
scale bar 200 nm

ameter and smaller particles with a diameter of 8 nm. It

could be hypothesised that as the plant grows and there is
increasing water movement, these separate particles could
aggregate more towards the outer region. As they agglomerate, these particles could influence the density fluctuation in the middle part.
Structurefunction relation

Fig. 4 Dense silica with pores or channels, probably caused by the

organic matrix; scale bar 30 nm

between are visible (Fig. 4). The holes seem to be the

cross section of fibres. These structures are caused by the
network of organics. The preferred orientation of the same
is along the radial and parallel axis of the plant. This indicates that the organic part associated with the silica might
be the template directing the localisation of silica particles.
Earlier experiments on silica deposition [20, 21] have shown
that it is a permeation and void filling process rather than
replacement of the cell walls, wherein the organic part
acts as template for the deposition. A mechanism for the
nucleation of amorphous silica on plant materials [20] involving hydrogen bonding between the hydroxyl groups
in the silicic acid and cellulose, lignin within the organic
tissue has also been proposed. The silica with the associated organic molecules could account for the mechanical
strength of the plant and the future work should address
the analysis of this organic part.
Towards the inner region a transition was found from
the dense structure to a more loose arrangement of separated particles (Fig. 5). These particles were principally of
two different sizes, larger particles of about 60 nm in di-

The structure of silica in various parts of the horsetail, as

presented above, appears to correlate with the function of
the corresponding part of the plant. The outer layer is the
one which is more prone to structural damage and utilised
in the water and other exchange activities of the plant.
Hence, the silica remains as a thin layer with not a very
compact arrangement. It could be that the smaller particles have been leached away, leaving behind the larger
particles which remain as the thin surface layer. In case of
the middle layer, the compact dense arrangement could aid
in the stiffness of the plant and thereby prevent the attack
from predators as well. The inner region with the small
loose silica particles could be the region where the dis-

Fig. 6 Typical amorphous diffraction pattern of silica made from

the denser structures (Fig. 4)

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solved silica is taken up from the soil with other elements,

is getting polymerised to form small particles initially.
These particles could later agglomerate and form granular
spheres and as water flows towards the ends, they get concentrated to form a dense arrangement as in the middle
and outer layers.

teristic for the amount of inner surfaces. Gerber et al. [23]

have found values between 15 nm1 in case of silica glass
up to 16.5 nm1 for silica gel grown at a pH value of 2.2.
In the present study, the value of 16.5 nm1 could correspond to a silica structure with high inner surfaces. Therefore we conclude that the silica with high inner surface in
horsetail is more similar to the ones in gel and this medium range order is not influenced by organic templates.

Electron diffraction studies

The electron diffraction pattern in Fig. 6 shows the silica
to be principally of amorphous form. On further analysis
of the scattering intensities, we have deduced the reduced
intensity function s*i(s) and show it in Fig. 7. From this,
the radial distribution function was determined and the interatomic distances estimated (Fig. 8). The first four interatomic distances in our sample are similar to silica structures from sources like vapour deposited silica, silica gel
and silica glass [22], with a comparatively higher ordering
than in vapour deposited silica but less than in silica glass.
The medium-range order can be described with the first peak
position in the reduced intensity function which is charac-

SAXS studies

Fig. 7 Reduced intensity function calculated from the diffraction

pattern in Fig. 6

Fig. 9 Double logarithmic plot of intensity of plasma ashed leaf of

Equisetum arvense

Fig. 8 Radial distribution function of silica in horse tail

Fig. 10 Plot of chord distribution function showing particles of

8 nm in diameter

SAXS measurements were done on leaf and stem parts of

Equisetum arvense. This is an integral method of structure
investigation and results from this method can confirm results from TEM measurements in the range of up to 30 nm.
The logarithmic intensity versus q plots of these natural
samples showed almost the same structure. In case of the
plasma ashed sample, the intensity was higher by more
than 10 times and the similarity in curves proved that the
whole structure was not altered during the ashing process.
As the curves were similar, a single curve is presented

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Fig. 11 EDX spectra of plasma ashed sample substantiating
the removal of organics

(Fig. 9) which is representative of all the samples. In the

double logarithmic plot, the initial part of the curve is linear followed by a small shoulder. This Guinier region corresponds with particle sizes of nearly 8 nm in diameter.
The chord distribution function calculation (Fig. 10) yields
particle sizes of nearly 8 nm in diameter. On curve fitting,
under the collimation conditions, the slope of the linear
region obtained is 3.2. This value in the double logarithmic plot can be correlated to the surface fractal with a
fractal dimension (Df) using the formula slope=6Df The
fractal dimension value obtained is about 2.8. We observed particles of this diameter in TEM investigation as
well but such particles seemed to have a loose arrangement. These two investigating methods can be correlated
in the way that the particles are connected at the surface
thereby having a fractal dimension value of nearly 2.8 and
a loose packing as well. But specific studies with SAXS
cannot be made on a particular section of the leaf or stem,
which is possible by TEM.
Plasma ashing
The silica deposits in plant cells cannot be easily studied
under the microscope without special treatment of the
plant material, either to destroy the organic matter or distinguish the silica from cell wall. Various methods have
already reported the removal of organics; however, it is
apparent that none of the methods were able to remove the
organics completely, and harsher treatments led to a damage of silica structure. The surface of horsetail is characterised by a well-defined micro morphology and the various surface structures have been described in detail by Perry
et al. [14]. A low-temperature plasma method [24, 25] of
obtaining ash to avoid thermal effects had been reported
earlier, but has not been widely used. The current samples, without any fixation, were plasma-ashed in an oxygen atmosphere. The highest power of 150 W for nearly
4.5 h led to the removal of almost all the organics. An EDX
spectrum of the plasma-ashed sample (Fig. 11) substantiates the same. As shown by the SEM photo in Fig. 12, this
method doesnot lead to significant structural damage and

Fig. 12 Undamaged stomata on the surface of a plasma ashed

sample; scale bar 20 m

the surface morphology of the plasma-ashed sample is

similar to that of the untreated sample. This shows that the
silica mimics the underlying cell structure thereby protecting the plant from predators and offering stability to
the plant as well.

Conclusions
From the elemental mapping and EDX, we can conclude
that the silica is concentrated on the surface as a thin
layer. Ultra structure studies with TEM reveal the thickness of the surface layer to vary from a few nanometers to
about 7 m. The silica layer in the outer part was more
dense due to closer packed particles. The middle layer
also has a dense silica structure with some holes or chan-

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nels in between and probably the organic part fills these

channels. Hence this surface layer could be described as a
compound structure of silica interwoven with the organics. Towards the inner part, a loose arrangement of silica
particles is found and this could be the region where the
silica accumulates. Electron diffraction shows an amorphous silica. The RDF is typical for connected SiO tetrahedrons forming a network as in a silica xerogel. The resulting network has high amount of inner surfaces as in a
dried gel produced at pH 2.2. The organic part has no effect on silica structure in the atomic range. From the intensity plot of SAXS measurements, the presence of surface fractal with a particle size of about 8 nm is deduced.
These particles are connected at the surface in a random way
and also correspond to the particle size deduced in TEM
investigations. Plasma ashing is one of the best methods
to remove the organics without significant surface destruction and this silica layer at the surface is dense offering
strength and protection to the plant.
Acknowledgement The authors would like to thank the DFG for
funding the project 12001361 under the head Schwerpunktsprogramm (SPP) 1117: Prinzipien der Biomineralisation.

References
1. Kroger N, Deutzmann R, Sumper M (1999) Science 286:1129
1132
2. Kroger N, Deutzmann R, Sumper M (2001) Biol Chem 276:
2606626070
3. Jones LHP, Handreck KA (1967) Silica in soils plants and animals. Adv Agron 19:107149

4. Kaufman PB, La Croix JD, Rosen JJ, Allard LF, Bigelow WC

(1972) Am J Bot 59:10181025
5. Perry CC, Mann S, Williams RJP (1984) Proc R Soc Lond B
222:427438
6. Perry CC, Williams RJP, Fry SC (1987) J Plant Physiol 126:
437448
7. Parry WD, Smithson F (1964) Ann Bot 28:169
8. Iler RK (1979) The chemistry of silica. Wiley, New York, p 741
9. Bonnett OT, University of Illinois at Urban-Champaign College of Agriculture Agricultural Experiment Station Bulletin 742
10. Tyler V (1993) In: The honest herbal, 3rd edn. Pharmaceutical
Products Press, New York
11. Cloutier D, Watson A (1985) Weed Sci 33:358365
12. Kaufman PB, Dayanandan P, Takeoka Y, Bigelow WC, Jones
JD, Iler R (1981) In: Silicon and siliceous structures in biological systems. Springer, Berlin Heidelberg New York, pp 409
449
13. Page CN (1972) New Phytol 71:355369
14. Perry CC, Fraser MA (1991) Phil Trans R Soc Lond B 334:
149157
15. Perry CC, Tucker TK (2000) J BioI Chem 5:537550
16. Dove DB (1973) In: Hass G, Thun RE (eds) Physics of thin
films. Academic Press, New York, p 7
17. Wright AC, Leadbetter AJ (1976) Phys Chem Solids 17(5):
122145
18. Cockayne JH, McKenzie DR (1988) Acta Cryst A 44:870878
19. Knoblich B, Gerber T (2001) J Non Cryst Solids 283:109113
20. Leo RF, Barghoorn ES (1976) Silicification of wood. Harvard
University Botanical Museum Leaflet 251-47
21. Harrison CC (1996) Phytochemistry 41:3742
22. Kamiya K, Dohkai T, Wada M, Hashimoto T, Matsuoka J,
Nasu H (1998) J Non Cryst Solids 240:202211
23. Gerber Th, Himmel B, Hbert C (1994) J Non Cryst Solids
175:160168
24. Umemoto K (1974) Yakugaku Zasshi 94:380386
25. Iler RK (1979) The chemistry of silica. Wiley, New York, p 741