How does the ROI affect the

thresholding?

Method note
MCT-024
2 Bruker microCT method note:Impact of a ROI on your analysis

Micro-computed tomography can be applied for the visualization of the inner

structure of a material or biological tissue in a non-destructive manner. Besides
visualization, image analysis is an important topic in micro-CT, to obtain
quantitative parameters from scanned datasets. SkyScan has developed analysis
software CTAn to serve this purpose to a large extend. An important issue to consider
in CTAn is the application of a region of interest (ROI). This application note will give
an example to illustrate the importance of this ROI and the correct use of it.

Introduction
CTAn performs quantitative measurements both of densitometry (voxel
attenuation coefficient or calibrated density) and of morphometry. The
morphometry image analysis is based on binary images. Binary images
contain only black and white pixels, representing the non-selected or
selected pixels respectively. The process of selecting pixels is called
binarization and in CTAn this is performed by means of segmentation or
thresholding. Simple global or adaptive methods allow for easy
segmentation.
Acquisition of quantitative data can be greatly influenced by defining the
region of interest. CTAn offers a number of tools for drawing ROI’s with a
wide range of shapes. The importance of this ROI needs to be emphasized.

Methodology
Let’s consider the following illustrative dataset, a sand sample. The dataset
has been acquired with a SkyScan1172 with a 10MP Hamamatsu camera.
The sample was scanned at 44kV with a 0.5 mm Al filter at 360° and
reconstructed with NRecon. This 8-bit reconstructed dataset is loaded in
CTAn and has 256 different grey values in the range [0,255].

Figure 1. Illustrative sand sample in CTAn.

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3 Bruker microCT method note:Impact of a ROI on your analysis

The table below (

Table 1) shows the different steps in 2 comparative methods. In the
first method an ROI is drawn, a new dataset is saved and reloaded
from this ROI, and 2 different thresholding values have been applied
to select the air in between the sand particles. The second method
follows the same steps, but there is an additional step in between. It
not only loads the new dataset from the ROI, but also the ROI itself.
Understanding the impact of this key step, and how this affects the
results (in step 4 and 5) is crucial for analysis.

Table 1. Comparison of 2 analysis methods

Method 1 Method 2
Step 1: draw an ROI Step 1: draw an ROI

STEP 2: save new dataset from STEP 2: save new dataset from
ROI , load (this) new dataset ROI , load (this) new dataset

STEP 3: Load the ROI (again)

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4 Bruker microCT method note:Impact of a ROI on your analysis

STEP 3: Apply thresholding STEP 4: Thresholding [1,25]

[1,25]

STEP 4: thresholding [0,25] STEP 5: thresholding [0,25]

Result & discussion

CTAn looks at an image as a square collection of pixels. When you are
loading an image with a different shape, CTAn will automatically generate
a square picture from this shape, by including a number of black pixels with
grey value of 0. When reloading the dataset in step 2, the round image has
now become square. When thresholding the range [1,25] these pixels are
not included, but when the boundary of the thresholding range is changed
to [0,25], these pixels are also binarized, showing that these pixels have a

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5 Bruker microCT method note:Impact of a ROI on your analysis

greyvalue of 0. In the second method, not only the dataset has been
reloaded, but also the ROI. This is a way of defining the image boundary.
The image is now round. The pixels in green are not part of the image,
which is seen when applying the thresholding ranges [1,25] and [0,25]. The
image is now defined within the boundaries of the ROI.
This difference has a consequence in analysis. When analysing the
percentage of air in this sand sample for example, one can be interested in
the object percentage. In the table below,
Table 2, this parameter is calculated for each of the four possible outcomes
from
Table 1. When applying a threshold of [1,25], the object percentage is
39.8%. When neglecting to reload the ROI, an artificially low value of 30.7%
is obtained. Although the absolute amount of white pixels remains the same
This value is lower because the total ROI area is larger. There are now less
white pixels with respect to the ROI. For a thresholding value of [0,25], the
object percentage would be 49.8%.. When the reloading of the ROI would
have been neglected, this would lead to an artificially high value of 61.3 %.
The pixels surrounding the round sand sample are now also included in the
image, and considered as part of the object, which is obviously a false
statement.

Table 2. analysis of different ROI & thresholding

Not reloading ROI Reloading ROI

ROI Area = 9.14 106 micron ² ROI Area = 7.06 106 micron ²

Object Object
[1,25]

%= %=
30.7% 39.8 %

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6 Bruker microCT method note:Impact of a ROI on your analysis

Object Object
[0,25]

%= %=
61.3% 49.8 %

Conclusion
In this application note the importance of understanding the ROI has been
pointed out. By means of an example dataset it has been shown that
neglecting to reload the ROI may result in an inappropriate amount of pixels
being falsely considered as part of the image, which can lead to incorrect
analysis results. Obtained values can be artificially high or low, depending
on the protocol that is used. In order to prevent this from happening, one
needs to carefully address this issue and design an appropriate ROI.

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