Appl. Phys.
A 66, S559–S563 (1998)
Surface structure of native cellulose microcrystals by AFM
A.A. Baker1 , W. Helbert2 , J. Sugiyama2 , M.J. Miles1
1 H.H. Wills Physics Laboratory, University of Bristol, Bristol, BS8 1TL, UK
(Fax: +44-117/925-5624, E-mail: andy.baker@bristol.ac.uk)
2 Wood Research Institute, Kyoto University, Uji Kyoto 611, Japan
Received: 25 July 1997/Accepted: 1 October 1997
Abstract. Atomic force microscopy (AFM) has been used
to study the surface of native cellulose I microcrystals from
Valonia ventricosa. High-resolution images show clear struc-
tural details of the surface, namely the 0.52 nm repeat along
the cellulose chains resulting from the glucose sub-unit and
the inter-molecular spacing of ∼ 0.6 nm. Cellulose from Val-
onia exists naturally in both a triclinic (Iα ) and a monoclinic
(Iβ ) crystal form within the same microfibril; the main differ-
ence between them being a displacement of adjacent chains
by a quarter of the c-axis period to give either a diagonally
shifted or staggered arrangement of the cellobiose units. The
most significant finding in this work is that it has been pos-
sible to image the cellobiose repeat along the chain because
of topographic differences associated with the asymmetric
glucose unit, and thus identify triclinic structure on the mi-
crocrystal surface. Computer modelling has been used to con-
struct pseudo-AFM topography images from Connolly sur-
faces of the facets of the two different crystal forms, and the
triclinic models are in excellent agreement with the real im-
ages obtained by AFM.
Cellulose is a homopolymer of β-[1,4] D-glucose molecules
linked in a linear chain, with alternating sub-units in crys-
talline cellulose rotated through 180◦ . Native cellulose I
microfibrils are highly ordered crystals and evidence from
13
C NMR spectroscopy and electron diffraction suggests that
they consist of both triclinic (Iα ) and monoclinic (Iβ ) allo-
morphs [1–4]. In both allomorphs, the molecules have very
close to strict two-fold screw symmetry along the chains,
which means that the true repeating unit is cellobiose, com-
prising two glucose units. The difference between them is
a shift of adjacent hydrogen bonded sheets along the chain
axis, resulting in either a stagger or a diagonal shift of the
cellobiose unit by a quarter of the c-axis period.
The cellulose used in this work is from the green alga Val-
onia ventricosa, consisting of Iα and Iβ phases roughly in the
ratio 65 : 35 [2]. It is known from studies of cellulose I from
Applied Physics A
Materials
Science & Processing
 Springer-Verlag 1998
Microdictyon that these two different crystal phases can co-
exist within a single microfibril [4]. One of the unresolved
problems of structural research into native cellulose I is pre-
cisely where the triclinic and monoclinic phases exist within
a microfibril [4].
We have applied the technique of atomic force mi-
croscopy (AFM) to image the surface of native cellulose I
microcrystals at ultra-high (sub-nanometer) resolution. The
AFM images under both propanol and water have clearly
shown the pitch of 0.52 nm along the molecule due to the
asymmetrical glucose unit and the inter-molecular spacing of
approximately 0.6 nm.
The topographical differences associated with the screw
symmetry along the cellulose chain have enabled us to iden-
tify the triclinic phase by direct imaging [5]. To support our
interpretation of the images, we have compared them with
computer-generated Connolly surfaces [6, 7] based on crys-
tallographic data from diffraction experiments [4, 8], assum-
ing no molecular relaxation at the surface compared to the
interior.
1 Materials and methods
Valonia ventricosa was harvested from a seabed in Florida
Keys, and the microcrystals were prepared by using the
method from Revol et al. [9]. Cellulose fragments were dis-
integrated in a homogeniser and sulphuric acid was added
slowly to improve the dispersion. The mixture was then heat-
ed to 70 ◦ C with strong stirring for 30 minutes. The resultant
dispersion was washed with distilled water and finally a drop
of chloroform was added before storage as a protectant.
Samples of Valonia for atomic force microscopy were
prepared by depositing a 5 µl drop of a diluted sample of
the cellulose (approximately 20 µg/ml) onto freshly cleaved
mica. For imaging under water, APTES-mica was used to im-
prove the binding of the cellulose to the mica substrate. The
APTES-mica was prepared by exposing freshly cleaved mica
to an atmosphere of aminopropyltriethoxysilane for several
hours [10].
S560

1.1 Atomic force microscopy

A NanoScope III controller with a MultiMode head (Digi-

tal Instruments, Santa Barbara, CA) was used to image the
cellulose crystals. Images were acquired by using the con-
tact mode under propanol or water, with both deflection (or
error signal [11]) and height signals recorded [5]. Unmodi-
fied, 200-µm-long silicon nitride cantilevers with a nominal
spring constant of 0.06 Nm−1 were used. Scan rates were
typically 10–20 lines per second, with the scan angle being
varied to obtain maximal contrast of the desired features. No
real-time filtering was applied and images are unprocessed,
except for flattening and plane-fitting where appropriate.

1.2 Connolly surfaces

Computer-generated models of the cellulose crystal structure

were used to study the cellulose surface. The monoclinic and

1 µm
triclinic unit cells were constructed according to the parame-
ters of Sugiyama et al. with atomic co-ordinates from electron
diffraction [4], which were further characterised by molecu-
lar dynamics simulations [8]. The crystal structures were built
Fig. 1. AFM topography image taken in contact mode under propanol show-
in Cerius2 TM (Molecular Simulations Incorporated). Crystal ing a typical field of Valonia cellulose microcrystals
surfaces were then cleaved from these bulk structures, and
the Connolly surface generation algorithm in the Cerius2 TM
program used to produce a surface [6, 7]. The size of the
probe rolling over the surface in the figures presented here
was 0.2 nm, comparable with the size of a water molecule.
2 nm
Larger probe sizes simply smoothed the surface and revealed
less detail, but did not obscure the key structural features of
the surface. The size of the probe is considerably smaller than
the 20–50 nm average radius of a typical AFM tip (Digital In-
struments specification), but the high resolution of the images
obtained is very likely to result from the presence of much
smaller micro-asperities at the end of the tip.
The generated surfaces were exported to custom-written
software to convert the surface co-ordinates into a pseudo-
AFM topography image.

2 Results and discussion

Figure 1 shows a typical low-resolution image of a sam-

ple of Valonia microcrystals imaged under propanol. The
long, straight microcrystals are evenly distributed and range
slightly in size. The measured heights and widths are in agree-
ment with the known size of 20 nm × 20 nm [12–14] if we
take into consideration the well-known effect of tip broad-
ening and the difficulty in making precise vertical measure-
ments [15, 16].
A high-resolution deflection image is shown in Fig. 2,
where parallel features orthogonal to the chain axis direction
(indicated by the arrowed line) can be clearly seen. The peak
intensity in the 2D Fourier transform shown in the corner of
the image is at 0.55 nm, which is in good agreement with the
diffraction measurements of the pitch along the chain due to
the glucose sub-unit. At this scan angle, the inter-molecular
spacing is not clearly resolved since it is features orthogonal
to the fast scan direction, which will be seen most clearly. In
deflection mode images, the high spatial frequency compo-
nents of the surface will be enhanced by the differentiation
Fig. 2. A deflection mode image of the cellulose surface taken under
propanol. The cellulose chains run parallel to the arrowed line on the im-
age. The spacing between the bands is 0.55 nm, in good agreement with
the known crystallographic spacing. The arrow on the 2D FFT of the image
data in the top corner highlights this spectral periodicity
effect as the feedback electronics respond to the gradient of
topographic features [5].
Figure 3a shows the most interesting structural details.
The cellulose chain axis runs almost vertically in the image,
and at least two regions can easily be seen where there is
a pattern of bright spots oriented diagonally at 64 ± 2◦ to the
chain axis. The line profile in Fig. 3b was taken from the

a 5 nm
b 1.09 nm
1 nm
Fig. 3. a A software zoom from a larger deflection mode image of the
cellulose surface taken under propanol. The molecular chains run almost
vertically in the image. Several regions can be seen where there is a pat-
tern of bright spots oriented diagonally at 64 ± 2◦ to the chain axis. The two
arrows indicate where the line profile shown in b was taken from. b Line
profile taken along the cellulose chain between the arrows shown in a, and
averaged across the width of the chain. The main bright features are spaced
at 1.09 nm, in close agreement with the cellobiose repeat interval. No ordi-
nate scale is shown since the line profile is taken across a deflection image,
and the grey-scale has no direct relationship to height. c Fourier transform
along the line profile highlighting the spectral peak at 1.09 nm
image between the two marker arrows, averaged across the
width of the chain. It should be noted that this is a line pro-
file from a deflection image, so the ordinate of the line profile
does not correspond to height changes and is presented in ar-
bitrary units. The prominent peaks are separated by 1.09 nm,
as is clearly shown in the Fourier transform of the line pro-
file in Fig. 3c. This pattern can easily be seen along the chains
adjacent to the one profiled. A similar analysis from a line
profile taken across the chains shows an inter-molecular pe-
riodicity of 0.56 nm, which is in good agreement with the
values found from diffraction (see Fig. 4).
This ∼ 1 nm spacing along the chain axis is extremely
significant. The cellulose chain has a two-fold screw sym-
metry because of the 180◦ rotation of the glucose sub-unit.
Thus although the glucose repeat is about 0.52 nm, the crys-
tallographic repeat of the cellobiose unit is 1.04 nm, and glu-
cose units alternately display the C2-C3 and O5-C5 faces.
The C5 atom on the glucose ring is bonded to a large hy-
droxymethyl group, whereas the C2 and C3 carbon atoms
bind smaller hydroxyl groups. The spatial arrangement of
these chemical groups must be such that the large hydrox-
ymethyl group on the O5-C5 face will be presented as a raised
topographic feature. It is this topographic difference which
S561
we believe is mainly responsible for the pattern illustrated
in Fig. 3a.
To compare our AFM results with the known crystal struc-
ture of native cellulose I, we used a computer modelling
package to generate Connolly surfaces [6, 7] of the cellulose
crystals. The Connolly surface is formed by rolling a probe
sphere over the crystal surface; if the rolling probe has zero
radius, then the van der Waals surface is generated. Our mod-
elling was simplistic in that no attempt was made to account
for other interaction forces and scanning parameters (such as
orientation, imaging force, and scanner hysteresis) which are
well known to be important in AFM.
In the introduction it was stated that two crystal forms
exist in native cellulose, one having a triclinic unit cell and
the other being monoclinic. Figure 4 shows the differences
between these two crystal forms as modelled at the cellu-
lose surface. The differences are immediately obvious; the
triclinic surfaces (Fig. 4a,b) show a diagonal shifting of the
bright spots, whilst the raised topographic features are stag-
gered on the monoclinic surfaces (Fig. 4c,d). The spacing of
the brightest spots along the chain is that of the cellobiose re-
peat (1.04 nm), as expected from the screw symmetry referred
to earlier in this discussion. The structural difference between
c these two phases is a shift of adjacent chains by a quarter
of the c-axis period, and the thermodynamically metastable
triclinic form can be converted into the monoclinic form by
annealing at high temperatures [17, 18].
Connolly surfaces have been used before to classify the
crystallographic face of the cellulose surface, although it was
Triclinic (100) Triclinic (010)
1.04 nm
63o 67o
0.53 nm 0.62 nm
(d010) (d100)
Monoclinic (110) Monoclinic (110)
1.04 nm
0.61 nm 0.54 nm
(d110) (d110)
Fig. 4. Artificial AFM topography images generated from Connolly surfaces
of the appropriate crystal faces. The cellulose chain axis is oriented verti-
cally in all of the images. The spacing between the highest points along the
chain is 1.04 nm in all of the images, corresponding to cellobiose interval
between prominent hydroxymethyl groups on the O5-C5 face of the glucose
ring. The inter-molecular d-spacings at the surface are shown for each of the
crystal faces
S562
necessary to make the comparisons in reciprocal space [19].
The similarity of the triclinic Connolly surfaces shown in
Fig. 4a,b with several areas of the image shown in Fig. 3a will
be apparent immediately. This pattern is not visible over the
entire image, possibly because of some disruption of the cel-
lulose surface. It is also difficult to achieve the highest quality
contrast over the entire image at this resolution because of
noise and scanning influences. As referred to in the introduc-
tion, cellulose from this source is likely to show other areas
of the surface with monoclinic character (although the tri-
clinic phase is dominant) and this is the subject of on-going
investigations. Although the AFM image shown is a deflec-
tion image, the contrast will primarily be due to topographic
features. Other influences are expected to be present such as
elastic deformation and frictional forces [20, 21], but these do
not detract from the fact that alternate sub-units are inverted
along the cellulose chain, which manifests at the crystal sur-
face as a topographic change.
Figure 5 supports this hypothesis further. This deflection
image of the Valonia surface was taken under water, with the
chain axis parallel to the dotted white box. A repeating feature
can be seen with some qualitative indication that the bright,
wide band is split in two, such that there is a “pairing” of sub-
a 2 nm
1.13 nm c 3. J. Sugiyama, T. Okano, H. Yamamoto, F. Horii: Macromolecules 23,
0.56 nm
1 nm
b
Fig. 5. a A deflection image of the cellulose surface taken under water.
The chain axis is parallel to the dotted white box which indicates where
the line profile was taken from in b. There is a qualitative indication that
the bright, wide bands are split into smaller sub-bands. In b, this band-
ing can be clearly seen along the line profile. The major repeat of 1.13 nm
(black arrows) is sub-divided by a smaller repeat of 0.56 nm (grey paired
arrows). This observation supports the idea that alternate glucose units are
exposing a more prominent chemical group on the surface and making the
cellobiose repeat more significant. c shows the Fourier transform along the
line profile in b
bands. The line profile shown in Fig. 5b, averaged across the
width of the dotted white box, again clearly shows the repeat
of about 1 nm between the most prominent peaks. Most of
these peaks are split into two, for example on the right-hand
extreme of the line profile. These larger and smaller inter-
vals appear prominently on the Fourier transform of the line
profile.
This again shows the effect of the two-fold screw sym-
metry along the chain. It is interesting that in Fig. 2 there is
apparently no increased significance given to the cellobiose
repeat. We have obtained many images of the cellulose sur-
face, some of which emphasise the cellobiose interval whilst
others do not seem to stress the difference. The interaction
between the tip and the surface, as well as the scanning and
feedback parameters are certainly very important in this re-
gard. The change of environment from propanol to water may
also be important; either because the chemical groups ex-
posed to the surface are surrounded by a different medium,
or because the tip–sample interaction is modified. Further ex-
periments are in progress to assess this.
3 Conclusions
We have applied atomic force microscopy to the study of the
surface of cellulose microcrystals and clearly resolved the
intra-chain periodicities of 0.52 nm and 1.04 nm due to the
glucose and cellobiose units respectively, as well as the spac-
ing between adjacent cellulose chains on the surface.
Most importantly, we have observed the cellobiose repeat
as a result of the bulky O6 group being detectable topo-
graphically. Direct comparison in real-space between AFM
deflection images and computer models of the cellulose sur-
face clearly demonstrate that the triclinic character known
in the crystal has been identified on the cellulose surface.
This real-space differentiation between the triclinic and mon-
oclinic allomorphs, which differ only by a displacement of
the cellulose chains along their axis by 0.26 nm, was achieved
without the need for filtering or averaging. To our knowledge
this is one of the highest resolution studies of a biological
sample at the present time.
Acknowledgements. AAB would like to thank the EPSRC and IACR-Long
Ashton for financial support.
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