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3D imaging of aerated emulsions using X-ray microtomography

Conference Paper · April 2011

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3D imaging of aerated emulsions using X-ray

microtomography
G. van Dalen, M.W. Koster

Unilever Research & Development, Advanced Measurement & Data Modelling,

Olivier van Noortlaan 120, NL-3133AT Vlaardingen, Gerard-van.Dalen
@unilever.com

Introduction
Stabilisation of air bubbles in liquid food systems is notoriously difficult. They have to
be stable during production or preparation. Examples of relative stable aerated
products are bread, ice cream and mousses in which the gas bubbles are entrapped
within the solid continuous phase. However liquid or soft solid foams are
thermodynamically unstable resulting in an increase in bubble size (coarsening) in
time and eventually to complete loss of air. These products are subject to
destabilisation processes such as coalescence and Ostwald ripening (diffusion of air
from small to large bubbles). Stabilisation of aerated soft solid oil in water (o/w)
emulsion offers a huge challenge.

A better and comprehensive insight into mechanisms responsible for the formation
and stabilization of foam structures is required. Quantitative methods are needed for
the characterization of aerated products and therefore suitable for the derivation of
substantiated structure-function relationships. The amount of gas and the bubble size
distribution of these products have to be followed in time. This paper shows how X-
ray microtomography (µCT)1,2 can be used for the 3D visualisation and quantitative
analysis of bubbles in aerated emulsions during storage.

Method
Model emulsions were imaged using a Skyscan 1172 desktop µCT system. The used
image acquisition parameters are listed in Table 1. Tomographic reconstructions
were performed on a cluster computer (HP 220c server Blade with quad core Xeon
CPU with 16 cores in total) using Nrecon (v 1.6.3.2). A smoothing factor of 4, beam
hardening correction of 50% and ring artefact reduction of 20 was used. The features
in the stacks of the 2D µCT images were identified and measured using an image
analysis toolbox (DIPlib from the Delft University of Technology). For visualisation in
3-D space, isosurface rendering was used (Avizo 6.2 from the Visualization Sciences
Group). Images of time series were aligned in Avizo using affine registration. Before
registration a coarse manual alignment was performed using marks on the sample
holder wall (small hole visible in Figure 14 ). The quantitative analysis of the bubble
size distribution will be reported in a separate paper3.

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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Table 2 μCT acquisition parameters

Tube Image size, Image pixel size, Kv - μA Rotation step Frame Number
o
diameter pixels μm (/180 ) averaging scans vertical
mm
6 4000*4000 2.0 59 -167 0.20 4 1
** 2.0 59 -167 0.23 2 1
4000*4000
11 4000*4000 4.0 59 -167 0.20 4 1
2000*2000
*,** 8.0 59 -167 0.23 2 2
26 3564*3564* 8.4 89 -112 0.20 4 3

* partial width used, **settings for storage trial

Figure 1 Projection image showing the total scanned area (obtained by stitching
multiple vertical overlapping scans).

Selection of an appropriate sub sample of the whole aerated product is one of

the most critical steps in the procedure. The sub-sample should be
representative for the total product. For this purpose plastic cylindrical sample
holders are used (Figure 2A). The upper part of the sample holder consist of a
removable open tube with a length of 20 mm. This tube was filled by pressing the
tube gently into the aerated product, preventing disturbance of air bubbles in the
sample and not to include additional air in to the holder. Rotation of the tube or
suction of the sample into the tube may cause deformation of the bubbles. To follow
air bubbles in time during a storage trial and to prevent subsampling at each time
point new sample holders were developed. These sample holders are made of
Perspex (polymethyl metacrylate, PMMA) and are sealed hermetically using O-rings
(Figure 2B). However during first trials, loss of the continuous phase due to
evaporation was observed. This in turn influenced the air bubbles in the sub-sample.
The evaporation is caused by the permeability of Perspex. Dependent on the relative
humidity, Perspex can absorb water up to 2.2%4. Evaporation can be prevented
wrapping the sample holders with several layers of Parafilm (a plastic paraffin film
produced by Pechiney Plastic Packaging Company, USA). Alternatively sample
holders of polypropylene (PP) can be used (Figure 3). PP sample holders with an
inner diameter of 26mm were made from standard 50 ml centrifuge tubes with screw
cap from Sarstedt (www.sarstedt.com). Tubes were cut at the 20ml mark and
mounted on a special made base (see Figure 2C).

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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Figure 2 Sample holders for imaging emulsions.

Figure 3 Influence of sample holder on the storage of 2.5 – 3.5 gram water (w) and
an emulsion (o/w) in sample holders with an inner diameter of 11mm.

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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Results
Representative cross sections of µCT images of an aerated o/w emulsion with 25%
oil imaged at a pixel size of 2.0, 4.0 and 8.4 μm using sample holders with an inner
diameter of 6, 11 and 26 mm are shown in Figure 1. The air bubbles are clearly
visible within the fat/protein/water matrix by their high grey value (low absorption
coefficient). These images show a broad distribution of bubble sizes including very
small and very large bubbles. A high magnification (pixel size of 2.0 μm) reveals the
presence of clusters of very small bubbles. However at this magnification the field of
view is limited, missing the very large bubbles. Selection of the size of a sample
holder will always be a compromise between obtaining maximum detail (resolution)
and being representative for the total sample. For accurate analysis of the total range
of bubble sizes in the sample shown in Figure 1, images could be made using
sample holders with an inner diameter of 6 and 26 mm. Larger sample holders will
also result in less sampling artefacts and will be less influenced by environmental
conditions during storage, representing more the actual shelf life.

Figure 1: µCT images of an aerated o/w emulsion imaged in sample holders with an
inner diameter of 6, 11 and 26 mm, scanned with a pixel resolution of 2.0,
4.0 and 8.4 μm, respectively. Bottom: enlarged view of the area indicated
by the yellow box.

Quantitative analysis of the size distribution of bubbles in μCT images requires

segmentation of the bubbles from the background (in this case the matrix is a
homogeneous emulsion). For segmentation using global thresholding, the grey
values of the matrix should be uniformly distributed over the total area within the
sample holder. However beam hardening will result in gradual change in grey level.
The edges of the sample appear more dark (higher absorption) than the centre
(cupping). Beam hardening is caused by the preferential absorption of low-energy
photons from the polychromatic X-ray spectra produced by the X-ray tube. A larger
sample holder and therefore a longer path-length through the sample results into

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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more beam hardening (Figure 4). Standard beam hardening correction5 during
tomographic reconstruction (square function in NRecon) reduced the cupping effect
but could not remove cupping completely. A better correction was obtained using a
3th order polynomial function (Figure 5B, C & D).

Figure 4 Relative grey level profilesi through reconstructed images of an o/w

emulsion without added air in sample holders with an inner diameter of 6,
11 and 26 mm showing the influence of beam hardening for different
sample diameters (with transmissionsii of 26, 17 and 15% and RSDiii
values of 24, 13 and 18% respectively at 50% beam hardening
correction). Images are shown without beam hardening correction.

Correction of beam hardening artefacts during reconstruction causes an increase in

noise, thus a decrease in quality (Figure 5A&B). An other possible strategy to reduce
beam hardening is to change the energy distribution of the X-ray beam. An increase
in tube voltage will increase the maximum photon energy of the continuous radiation.
The transmission through the sample increases linearly with the tube voltage (Figure
6B). However this has no significant influence on beam hardening and noise (Figure
6A). The lower energy part of the continuous radiation can be blocked by using
attenuation filters after or before it passes through the scanned sample. In this way a
harder, more monochromatic beam is presented at the sample. As can be seen from
Figure 7B, beam hardening can be reduced by using an aluminum filter of 0.5 mm. In
this case a beam hardening correction of only 50% will result in a flat profile. The

i The images and graphs were scaled relative to the average grey level in the centre. The
profiles show the vertical average over 50 pixels along a line shown in the images.
ii Transmission through the centre of the sample.
iii The relative standard deviation (RSD) of the mean grey level (matrix – air) over an area of
200*200 pixels.

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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main drawback of using a filter is that it degrades the X-ray signal at all energies to
some degrees, leading to greater image noise. Even when using a higher voltage
(59>95kV) and a higher acquisition (exposure) time (589>1767ms), a much higher
noise level is observed (Figure 7A). The longer acquisition time leads to an increase
in total scan time from 1.5 to 2.5 hours (scan with a rotation step of 0.20o/180o and
frame averaging of 4). So this will not be a preferred option, especially because
multiple scans (oversized scan) are needed to image the total sample holder.
100.0 25.0
A 80.0 25.0
B 90.0 Cupping
70.0
80.0 % RSD 20.0
20.0
60.0

cupping, %
70.0
cupping, %

50.0 60.0 15.0

15.0

% RSD
% RSD
40.0 50.0

10.0 40.0 10.0

30.0
30.0
20.0
Cupping 5.0 20.0 5.0
10.0 10.0
% RSD
0.0 0.0 0.0 0.0
0 10 20 30 40 50 60 70 80 90 100 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Beam Correction
hardening correction,
depth, % % Beam hardening correction,
Coefficient 3e order coefficient
a3
0 - second order third order
Beam hardening correction

C D
3e-order coefficient:
2.0
0.0 – 0.3 – 1.0
(0=100% BHC)
relative grey level

1.5
11 mm
1.0

0.5

0.0
0 500 1000 1500 2000 2500 3000 3500

profile, pixels

Figure 5 Influence of beam hardening correction (BHC) on cuppingiv and RSD of an

o/w emulsion without air in a sample holder with an inner diameter of
11mm. A: standard BHC ranging from 0% (no correction = linear function)
to 100% (square function), B: BHC using third order function 0 +
1.0*Intensity2 + a3*Intensity3), C: relative grey level profiles.

A B

Figure 6 Influence of voltage (power = 10W) on cupping and RSD of an o/w

emulsion without air in a sample holder with an inner diameter of 11mm.

iv
The cupping is calculated as a percentage of the average grey value at the border of the
sample relative to the average grey value at the centre.

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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A B C
0% - 50% - 100% beam
hardening correction

11 mm

Beam hardening correction, % Profile, pixels

Figure 7 Influence of beam hardening correction on cupping and RSD of an o/w
emulsion without air in an 11mm sample holder. Beam hardening was
reduced using an aluminium filter of 0.5 mm. Transmission = 55%.

Beam hardening can also be reduced using post processing of the horizontal cross
sections obtained after tomographic reconstruction. Two options were tested, using a
cross sectional average of the total stack of slices of 1: the sample (same dataset)
and 2: a physical phantom (separate dataset). The phantom should be uniform and
comparable density, size and shape, imaged using identical acquisition parameters.
As phantom an emulsion without air was used. For both methods the extent of the
beam hardening has to be same for each slice. For an aerated emulsion the beam
hardening effect is not significantly influenced by the vertical position.

The result of the first correction method is shown in Figure 8-1. The cross sectional
average was obtained by averaging a selection of about 40 slices, equally divided
over the total stack. These slices were first aligned and clipped at about 90% of the
average grey level of the matrix in the centre of the sample holder to reduce the
contribution of the low grey value of the bubbles. The cross sectional average was
blurred using a Gaussian filter with a sigma of 6 pixels and stretched between 1 and
99% percentile. After angular averaging this image was used to correct the total
stack of slices of the sample: each slice was divided by the correction image and
stretched between 1 and 99%.
The result of the second correction method using a non aerated emulsion as
phantom is shown in Figure 8-2. The procedure is comparable to the first method.
However in this case no clipping was used and the corrected image was aligned with
the image of the sample. The final results of both methods are more or less
comparable. Only method 2 also corrects for some residual ring artefacts (no blurring
used as in method 1).

For quantitative analysis the μCT images have to be smoothed to remove

noise. This can be done during tomographic reconstruction using Nrecon or
after reconstruction using image analysis software. Figure 9 shows the
influence of smoothing in Nrecon (smoothing kernel = asymmetrical boxcar)
on the visible quality of the images and on the obtained binary images. A low
smoothing factor results in binary images containing many small features and
large bubbles which are not filled. A large smoothing factor will decrease the
resolution, removing small bubbles. The impact of smoothing on small
bubbles is shown in Figure 10 and Figure 11. Bubbles with a diameter of 12
and 17 μm will disappear after using a smoothing factor of 4 and 8,
respectively. The selection of the smoothing factor will be a compromise
between accurate quantitative analysis of large and small bubbles. For this
application a smoothing factor of 4 was selected.

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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Profile, pixels

Figure 8 Correction for beam hardening using cross sectional averages of the total
stack of slices of the sample (top) and a non aerated emulsion (bottom).
The images were stretched between 1-99% percentile (BHC=50%).

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
 P a g e | 29

Figure 9 Influence of smoothing on the reconstructed image of an o/w emulsion

imaged in a sample holder with an inner diameter of 11mm. Grey level
image (1.7mm*1.7mm) stretched at 1% percentile with binary image in red
obtained using a fixed threshold value of 140.

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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Figure 10 Relative grey level profiles through an air bubble with a diameter of 17 μm
(FWHM) within a reconstructed image of an o/w emulsion in sample
holders with an inner diameter of 11 mm showing the influence of
smoothing during reconstruction. Grey levels were scaled between matrix
(1) and air outside the sample holder (0). Image pixels size = 4 μm.

Figure 11 Influence of smoothing on the contrast (relative grey level(matrix-bubble)/(matrix-

air)) and noise (RSD of the matrix) within a reconstructed image of an o/w
emulsion (Image pixels size = 4 μm).

The stability of aerated o/w emulsions was tested by imaging for each sample exactly
the same sub sample in time. For this purpose sub samples were imaged using
Perspex sample holders with an inner diameter of 6 and 11mm (Figure 2B). The
sample holders were stored at ambient conditions and imaged during 16 weeks.
Important microstructural parameters of aerated products are the air content and the
bubble size distribution. For this study the volume weighted mean diameter D[4, 3],
and the surface area weighted mean diameter D[3, 2] were measured.

Examples are shown in Figure 12 and Figure 13. The gas bubbles of this sample
were not stable during storage. The number of air bubbles per volume was reduced
and larger air bubbles were formed in time. For large bubbles coalescence was
observed (bubbles combine into larger ones). For small bubbles Ostwald ripening
was observed (Figure 14 and Figure 15). The air phase is transferred from small to
large bubbles due to their different Laplace pressures. It is controlled by the solubility

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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of air in the emulsion. At locations were the bubbles disappeared a higher absorption
was observed (Figure 16). This can be due to the migration of water (higher
absorption than oil/water).

Beside a decrease of the total volume of gas bubbles in time, also a decrease of the
emulsion volume was observed (Figure 13). This was caused by evaporation (see
discussion about sample holders in the method section).

Figure 12 μCT images of horizontal (middle) and vertical (bottom) cross sections of
an aerated o/w emulsion during 16 weeks storage in a sample holder with
an inner diameter of 11mm. The top row shows the 3D visualisation of the
bubbles (box size = 13.8mmx13.8mmx20.4mm, pixel size = 8.0 μm).

Figure 13 Bubble parameters of an aerated o/w emulsion during storage calculated

from the images shown in Figure 12.

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
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6 mm
1 day 3 days 1 week 1 month
Figure 14 μCT images of horizontal cross sections of an aerated o/w emulsion
during 1 month storage in a sample holder with an inner diameter of 6mm.
Enlarged views (2.4mm * 4.8mm) at different storage times with outlines
of bubbles detected at T0 in red.

Figure 15 3D visualisation of μCT images of horizontal cross sections of an aerated

o/w emulsion during 3 days storage with outlines (red grid) of bubbles
detected at T0.

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven
 P a g e | 33

xz

xy
1 day

xz
3 days xy

Figure 16 μCT images of horizontal and vertical cross sections (2.0mm * 2.0mm) of
an aerated o/w emulsion at 1 and 3 days after preparation.

Conclusion
X-ray microtomography allows in-situ observation and analysis of bubbles during
storage of aerated emulsions. Various coarsening mechanism can be identified and
investigated, namely coalescence and Ostwald ripening.

References:

1 Dalen G van, Don A, Nootenboom P and Blonk JCG (2009) Determination of

bubbles in foods by X-ray microtomography and image analysis, SkyScan user
meeting, Ghent, Belgium, 22 – 24 April 2009, 15-23
2 Dalen G van, Nootenboom P, Vliet LJ van, Voortman L and Esveld E (2007) 3D
imaging, analysis and modelling of porous cereal products using x-ray
microtomography, Image Anal Stereol 26: 169-177
3 Dalen G van and Koster, M. (2011) 3D visualisation and quantification of bubbles
in emulsions using μCT and image analysis, Proceedings of the 13th International
Congress of Stereology (ICS-13), Beijing, China, 19-23 October 2011
4 Properties of Perspex, Perspex South Africa (Pty) Ltd
http://www.perspex.co.za/Uploads/TechnicalManual/46'PERSPEX'%20PROPERTIES.pdf
http://www.perspex.co.za/Uploads/TechnicalManual/203XRAY%20TRANSMISSION.pdf
5 Casteele, E (2004) Model-based approach for beam hardening correction and
resolution measurements in microtomography, thesis University of Antwerp,
Belgium

G. van Dalen e.a., μCT imaging of aerated emulsions, SkyScan user meeting, April 13-15, 2011, Leuven

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