Segmentation of Blood Images Using Morphological Operators

t
Cecilia Di Ruberto , Andrew Dempster*, Shahid KhanH, Bill Jarratt
t
Dept. of Mathematics, University ofCagliari, Cagliari, Italy
*Dept. of Electronic Systems, University of Westminster, London, UK
ttNationallnstitute of Medical Research, London, UK
dirubert@vaxcal.unica.it, dempsta@cmsa.westminster.ac.uk

Abstract time-consuming so our task is to automate the process. A

major requirement is an efficient method to segment cell
This work describes a part of a malarial image images. This paper introduces a morphological approach
processing system for detecting and classifying malaria to cell image segmentation improving the accuracy of the
parasites in images of Giemsa stained blood slides in classical ·watershed-based algorithm.
order to evaluate the parasitaemia of the blood. A major
requirement of the system is an efficient method to 2. Morphological Granulometry and
segment cell images. This paper introduces a Gradient
morphological approach to cell image segmentation more
accurate than the classical watershed-hased algorithm. Mathematical morphology offers a powerful tool for
We have applied grey scale granulometries based on extracting image components that are useful in the
opening with disk-shaped elements, flat and non-flat. We representation and description of region shape, such as
have used a non-f/at disk-shaped structuring element to boundaries, skeletons, texture. We are also interested in
enhance the roundness and the compactness of the red morphological techniques for pre- or post-processing,
cells improving the accuracy of the classical watershed such as morphological filtering, thinning, gradient.
algorithm, while we have used a flat disk-shaped Granulometries were introduced fIrst by Matheron [4]
structuring element to separate overlapping cel/s. These as tools to extract size distribution from binary images.
methods make use of knowledge of the red blood cell By performing a series of morphological openings with
structure that is not used in existing watershed-based increasing structuring element size. we can obtain the
algorithms. granulometry function which maps each structuring
element size to the number of image pixels removed
1. Introduction during the opening operation with the corresponding
structuring element. Three kinds of granulometric
Generally i n blood analysis, doctors look for three function are currently used:
different kinds of cells, red, white and blood platelets.
These are distinguished by their dimensions and their 1. Number of particles of Ys. (f)
colour. In malarial blood the red corpuscles of vertebrates
2. Surface area of r s, (f) • often called size distribution
are infected by malaria parasites. Plasmodium, the
protozoan parasite that causes malaria, exists in a variety 3. Loss of surface area between r s, (f) and r5,+\ (f).
of different forms [8], which have successfully adapted to called pattern spectrum
different cellular environments, in both the vertebrate host
and the mosquito vector. The parasite develops in a highly
where ris the binary morphological opening,j represents
regulated manner through distinct cycles in the vertebrate
the original image. and Sk' k = 1.2. . .. . is a sequence of
host. In malarial blood we have to look for cells, red both
mature and young or reticulocytes and white. and for opening structuring elements of increasing size.

parasites. in different stages of life. immature and mature Due to the duality between openings and closings. the

trophozoites, schizonts and gametocytes. size distribution of the complement of an image can be
The aim of our system is to detect the parasites using a obtained by performing closings of the input image with

scan of a colour photograph of stained malarial blood the same family of structuring elements.

from a microscope in order to evaluate the parasitaemia of For our analysis we have chosen the pattern spectrum
the blood i.e. the number of parasites per number of red of the image as granulometric function. A large impulse
blood cells [1]. A manual analysis of slides is tiring and

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in the pattern spectrum at a given scale indicates the
presence of many image structures at that scale.
The concept of granulometries can easily be extended
to grey scale images by replacing the binary opening with
a grey scale morphological opening. In this case, the
measure used to define the pattern spectrum is the volume
instead of the surface area. In addition, different types of
granulometric curves can be computed, depending on the
underlying family of openings or closings used. For
example, curves based on openings with line structures
capture information on bright linear image features,
whereas curves based on closing with disk-shaped
elements capture information on dark, blobby image
parts. To capture information only on bright blobby image
parts (cells and parasites nuclei), we use grey scale
granulometries based on openings with disk-shaped
elements.
We use the morphological gradient of the image to
enhance the boundaries of the cells in order to isolate the
red cells from the background and to separate the
overlapping ones.
The morphological gradient of an image is defined in
terms of dilation (5 and erosion E, denoted p:
The grey scale morphological gradient highlights sharp
grey level transitions in the input image. As opposed to
gradients obtained using such methods such as a Sobel
operation, morphological gradients obtained
symmetrical structuring elements tend to depend less on
edge directionality.
3. A Morphological Approach to Segment
Blood Cells
Mathematical morphology is well suited to biological
and medical image analysis. Our proposed system is
based on morphological techniques, using granulometries
to evaluate the size of the red cells and the size of the
nuclei of parasites, opening or closing to enhance,
suppress or smooth some areas, thinning to improve cells
contours.
Before segmenting the image, the first steps in the
whole analysis procedure are the detection of white blood
cells and schizonts and their removal from the image to
segment [1]. We can ignore platelets in this analysis
because they are regarded as noise. The image produced
after segmentation contains two kinds of objects, "red
blood cell" and "background", with the aim to isolate each
individual red blood cell, especially when they are
overlapping and partially occluded and form clusters in
the viewing field of the microscope. In this image the cell
contours will be located and recognised.
The ideal image for segmentation would contain
compact cells on uniform background. The real images
contain noise from the sample, from the microscope light
or from the scanner. To isolate the red blood cells we use
the green component image because it is cleanest.
However, thresholding the input image, we see that cells
disappear into the background at the sides as noise comes
up in the centre (see Figure 1). Therefore we correct this
non-uniform illumination using a paraboloid model of the
illumination.
The noise is resolved performing a median filtering
using a 3x3 window. Thresholding the green image at this
stage would leave 'holes' in the middle of the red blood
cells. After filling the holes in the red cells by a
morphological area opening (see Figure 3), the last step of
this pre-segmentation phase consists in making the
background flat and clean. So, we threshold the image,
setting to zero all the pixels belonging to the background,
and remove items smaller than a red blood cell from it by
means of a morphological area-open filter (see Figure 4).
At this point segmenting the image means retrieving the
red cell blocks.
In order to identify red cell bodies we apply a
granulometric analysis on the image with nat background.
Morphological granulometry is a field that, among other
things, deals with determining the size distribution of
particles in an image. Figure 4 shows an image consisting
of objects of two main different sizes, cells and
trophozoites. Some objects are overlapping and they also
are too cluttered to enable detection of individual
using
particles. Because the particles we are looking for are
bright with respect to the background, the following
morphological approach can be used to determine size
distribution. Opening operations with structuring elements
of increasing size are performed on the original image.
The difference between the original image and its opening
is computed after each pass with a different structuring
element is completed. At the end of the process, these
differences are normalised and then used to construct a
histogram of particle size distribution. This approach is
based on the idea that the opening operation of a
particular size of structuring element has the most effect
on regions of the input image that contain particles of that
size. Thus a measure of the relative number of such
particles is obtained computing the difference between the
input and output image. Figure 5 shows the resulting size
distribution in our case. The histogram indicates the
presence of two predominant particle sizes in the input
image, relative to the nuclei of the trophozoites (3-7
pixels) and to the red blood cells (15-25 pixels).
So, the first step of the segmentation process consists
in applying a morphological granulometry by disk-shaped
structuring elements. This analysis provides information
about the size of the red cells. We estimate the size
distribution to evaluate the smallest size s of the red cells
(see Figure 5) and apply a morphological opening by a
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non fla t di sk-shaped struct uring element of size s on the 4. Conclusion
image. The morphological gra di en t on the result of the
o pening p roduces a first rough localisation of the re d cell In this paper we have presented a morphological
co ntou rs . W e threshold the g ra di en t i mag e and close the system to segment blood images . In Figures 1-10 the
holes in order to get a b i nary i mage we can use as marker different steps of th e procedure are showed. In the sample
i mag e in the cl as si cal water she d-ba sed s egmenta tion (see image, scanned from a colour photograph of stained
Figur e 6). In other words, we apply the classical malarial blood, there exist different kinds of parasites, a
watershed algorithm [9], cons train ed by the binary marker white polymorph blood cell, red cells both single and
image to find the contours of the red blood cells, at this connected. In Figure 11 the result of the classical
phase single and composite (see Fi gure 7). This gives us a watershed-based algorithm is pr esen te d .
partial segmentation with some com po un d cells still to We have applied grey scale granulometries based on
deal with. opening with disk-shaped elements, flat and non-flat. We
In fact the red blood cells can be o verlapping or have used a non-fla t di sk -shaped s tr ucturin g element to
partially occluded. Therefore each cell body area detected enhance the roundness and the compactness of the red
by segmentation can identify both individual and cells improving the accuracy of the classical watershed
composite cells.So cell para meters meas uring the algorithm, while we have used a flat disk-shaped
roundness are calculated. If a cell has a large ratio of the structuring element to that is not used in existing
maj or axis over the minor axis, i.e. it is elongated, it is watershed-based algorithms to separate overlapping cells.
treated as a composite cell (see Figure 8). The co mposite These methods make use of knowledge of the RBC
cells are then separated in the individual contrib uting stru cture that is not used in existing watershed-based
cells. al gori thm s.
In order to do that we apply a morpholo gic al opening
by a flat disk-shaped structuring element of si ze s on the
5. References
composite cells. The m orpholo gic al gr adient on the result
of the opening produces a first rough localisation of the (1] Dempster, A., Di Ruberto, e., "Morphological Processing
individual red cell bodies: thre shol din g the gradient image of Malarial Slide Images", Matlab DSP Conference 1999, nov.
produces rough individual contours (see Fig ure 9). After 16-171999, Espoo, Finland.
closing the small hole s betw een adja cent contours by a
morpholo gi ca l area closing, a morphol og ic al thinning [2] Gonzalez, R.e., Woods, R.e., Digital im age processing,
leaves contours around the individual cells (see Figure Addison-Wesley. 1993.
10).
[3) Harms, H., Gunzer, u., Aus, H.M., "Combined local colour
and texture analysis of stai ned cells", Compute,. Vision.
The gr anulometri c analy si s and the op enin g by non­
Graphics and Image Processing, 33, 364-376, 1986.
fl at , (hemisphere) shaped structuring element are the main
steps of th e segmentation process. Granulome try is used (4) Matheron, G., Randoms sets and integral equation, New
to capture information about objects of particular size and York, Wiley, 1978.
shape. In our cas e the objects of interests are cells, i.e.
bri ght blobby i mag e parts. So we use grey scale (5) Parthenis, K., Metaxaki-Kossionides, e., "Blood analysis
gr an ulo metries based on open ing w ith dis k -s haped using black and white digital images", J. Biomedical Eng.,
vo1.14, July, 287-292, 1992.
elements, flat and non-flat. We use a non-flat
(he mis ph ere) di sk -s ha ped structuring element to enhance
(6) Racky, J., Pandit, M., "Automatic generation of
the roundness and the compactness of the red cells before
morphological opening-closing sequences fot texture
applying the watershed algorithm as these features could segmentation", Proceedings of NSIP'99, Antalya, Turkey, 876-
be lost or be too weak to produce an accurate 880, 1999.
segme ntat io n if directly evaluated on the input image (see
Figure 11). On the contrary we use a nat disk-shaped [7) SOC Morphology Toolbox for Matlab 5, User's Guide,
structuring element to detect the points of contact in SDC Information System, 1999.
co mposite cells. Openings with flat disks capture
information about the height of overlapping objects, [8] Smyth, J.D., Introduction to animal parasitology,
allowing the separation of composite structures into the Cambridge, Cambridge University Press, 1994.
in di v idual composing parts.

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Figure 1. The initial sample image Figure 2. The WBC and the Figure 3. The result of filtering
schizonts detected and replaced and filling the holes
with the "background"

20 25 30 - - .

Figure 4. The RBCs on the flat Figure 5. The size distribution of Figure 6. The marker image used
background the RBCs in the segmentation

Figure 7. The contours of the Figure 8. The contours of the Figure 9. The gradient image after
RBCs, single and composite composite RBCs opening the composite RBCs with
a flat disk structuring element

Figure 10. The contours of the Figure 11. The result of the classical
individual RBCs watershed-based a lgorithm on
image in Figure 1.

[9J Soille, P., Morphological image analysis, Principles
and applications, Springer, 1999.
[1OJ Serra, J., Image analysis and mathematical
morphology, vol. 2, Academic Press, 1992.
[ll]Volkova, S.E., Ilyasova, N.Yu., Ustinov, A.V.,
Khramov, A.G., "Methods for analysing the images of
blood preparations", Optics & Laser Technology, vol. 27,
no. 4, 255-261, 1995.

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