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The project is developed on the MATLAB 7.0 The most straightforward and common of the
platform. pixel-based methods is thresholding in which all
pixels having intensity values above, or below,
some level are classified as part of the segment.
Thresholding is an integral part of converting an
2. METHODOLOGY intensity image to
a binary image. Thresholding is usually quite
As stated above, the study has three phases: fast and can be done in real time allowing for
1. Segmentation of individual cells from cell interactive setting of the threshold. The basic
image; concept of thresholding can be extended to
2. Mapping image of individual cell to an include both upper and lower boundaries
intensity image; and (slicing).
3. Use of GA to locate the global maxima
A major concern in pixel-based methods is
setting the thresholding or slicing level(s)
Let us start of with cell segmentation. appropriately. Usually these levels are set by the
In segmentation, the inputs are images but the program, although in some situations they can
outputs are attributes extracted from those be set interactively by the user. Finding an
images i.e. segmentation subdivides an image appropriate threshold level can be aided by a
into its constituent regions or objects. In the plot of pixel intensity distribution over the
study, three approaches to cell segmentation are whole image, regardless of whether you adjust
used. We shall see them individually in detail. the pixel level interactively or automatically.
Such a plot is termed as the Intensity
The first step in cell segmentation is loading the Histogram. Intensity histograms can be very
image and converting it to gray scale from the helpful in selecting threshold levels, not only
usual RGB color format. The image is then for the original image, but for images produced
further pre-processed. This step is common to by various segmentation algorithms. Initially
all the methods used. This step can be referred histograms can be useful in evaluating the
to as the Loading Subroutine. efficacy of different processing schemes: as the
separation between structures improves,
Loading Subroutine: histogram peaks should become more
distinctive. If the intensity histogram is, or can
a) Load the image be assumed as bimodal (or multi-modal), a
b) Perform grayscaling. common strategy is to search for low points, or
c) Improve contrast for the grayscaled minima, in the histogram.
image.
d) Improve precision (of the image from Keeping this in mind, there are two approaches
step c). based on which Approach 1 is designed and
implemented.
It may be remembered that the images are all in
the bitmap format (.bmp). One inclusion from The first approach is to improve the
the slide was pasted on a black background. determination of histogram minima is based on
This also reduces the effect of noise. All the observation that many boundary points carry
segmentation techniques are implemented on values intermediate to either side of the
this image to show variance in effectiveness of boundary. These intermediate values will be
the three methods. associated with the region between the actual
boundary values and may mask the optimal
threshold value. However, these intermediate
points have the highest gradient, and it should
be possible to identify them using a gradient-
sensitive filter, such as the Sobel or Canny on the final segmented image in order to plot
filter. After these boundary points are identified, the intensity graphs.
they can be eliminated from the image, and a Figure 1.1 displays broadly the various image
new histogram is computed with a distribution transformations of Pixel-based segmentation
that is possibly more definitive. Thus, this leads approach used for Approach 1. The original
to slightly better segmentation of the cell. image as shown in Fig 1.1 a) is first converted
to an increased contrast, high precision gray
The second threshold strategy is one that does scale image as shown in Fig 1.1 b). Firstly, edge
not use the histogram is based on the concept of pixels are removed and then the image is
minimizing the variance between presumed thresholded to give the final segmented image
foreground and the background elements. shown in Fig 1.1 c). Intensity plot are
Although the approach assumes two different determined on Fig 1.1 c) which gives rise to the
gray levels, it works well even when the intensity plot as shown in Fig 1.1 d) which is a
distribution is not bimodal. The approach uses meshed image intensity plot.
an iterative process to find a threshold that
minimizes the variance between the intensity 2.2 Approach 2: Image Segmentation
values on either side of the threshold level Using Multi-Thresholding Technique
(Outso’s method). This can be referred to as
Minimal Variance Iterative Technique. (Using AND - OR Operators)
The above two approaches are thus incorporated The results of several different segmentation
and implemented for Approach 1 as follows: approaches can be combined either by adding
the images together or more commonly, by first
1. Load the image of the cell and perform the thresholding images into separate binary images
necessary pre-processing like gray scaling and then combining them using logical
on the image (Loading Subroutine) operations. Either the AND or OR operator
2. Display images after pre-processing along would be used depending on the characteristics
with the intensity histogram of each segmentation procedure. If each
3. Remove the edge pixels from the image and procedure identified all of the segments, but
display the histogram of this modified also included non-desired areas, the AND
image. operator could be used to reduce the artifacts.
4. Determine thresholds using minimal Alternatively, if each procedure identified some
variance iterative technique described portion of the segment(s), then the OR operator
above could be used to combine the various portions.
5. Apply this approach to threshold both
images. This approach is used in Approach 2 where first
6. Display the resultant thresholded images. two, then three, thresholded images are
7. Perform class conversion of the final combined to improve segment identification.
segmented image The structure of interest is a cell which is shown
8. Plot the image intensity plots on a gray background. Threshold levels above
and below the gray background are combined
To remove the edge boundaries, first (after one is inverted) to provide improved
identify these boundaries using an edge isolation. Including a third binary image
detection scheme. While any of the edge obtained by thresholding a texture image further
detection filters can be used, for our approach, improves the identification.
Canny filter is used as it is more robust to noise.
The implementation of this filter will produce a Implementation of Approach 2:
binary image of the boundaries. This boundary
image is converted to a boundary mask by 1. Load the image of the cell and perform the
inverting the image. After inversion, the edge necessary pre-processing like gray scaling
pixels will be zero while all other pixels will be on the image (Loading Subroutine)
one. Multiplying the original image by the 2. Display images after pre-processing
boundary mask will produce an image in which 3. Perform linear filtering of the image using
the boundary points are removed (i.e. set to zero Low Pass Filter
or black). Perform class conversion (to double - 4. Threshold the image its complement
double-precision floating-point number array) 5. Threshold texture image
6. Combine the thresholded image and its
complement
7. Combine the above image with the
threshold texture image
8. Display thresholded and combined images
9. Perform class conversion of the final
segmented image
Fig 1.1 a) Original Image 10. Plot the image intensity plots
The results for the study are described in the Fig 4.1 show the intensity plots after each
form on the images obtained from the opening operation. Fig 4.2 shows the various
experiment. Image output obtained from all the intensity plots for the final thresholded image in
three approaches are displayed in a sequential Fig 4 (j).
flow chart format for easier understanding. The
study was carried out on an Intel Pentium 4, 4. DISCUSSION
3.20GHz, 512 MB DDR RAM. Experiments
were performed on MATLAB 7.0. In this project, an effort was made to study three
different segmentation approaches even though
As shown in Fig 2 for Pixel-based edge no broad comparison study was made. As it can
removed thresholding, after the initial pre- be seen, the output segmented images of all the
processing of the images, using a Canny filter, three methods are completely different. This
the edges are removed (Fig 2 g). This image is shows how segmentation results from different
then thresholded to give the final segmented approaches can yield to completely different
that pixel based methods are particularly
Fig 1.3 e) Second Opening
Fig 1.3a) Original Image
h) Segmented Image:
Thresholded Edge
d) High Precision
Removed
i) Intensity Plot (Mesh)
e) Gaussian Low
g) Edge Removed f) 3-D Convolution
Filter (Canny) Pass Filter
Fig 2.1 Various Plots of Image Intensity for Pixel Based Edge-Removed Thresholding
5.1.3 Histograms
Fig 2.2: Histograms of Gray Scaled Image and High Precision Image. There is
significant improvement in the histogram of High precision Image
Fig 2.3: Images of cell with (upper) and without (lower) intermediate boundaries
removed. The associated histograms (right side) show improved separability when
boundaries are eliminated
5.2 Multi-Thresholding
e) Thresholded f) Complement
Image of Threshold
e) Erode - I
The above figure shows various intensity plots after the use of “Opening Operation” before and after
thresholding of the image. The marked change in the plots can be seen. The plots before thresholding suffer
from presence of noise in the image. After thresholding, presence of noise seen is minimal.
5.3.3 Intensity Plots