Before to proceed with quantitative analysis of microCT images with our software VDL it is important to make an image pre-processing in order to increase the quality and reduce the quantitative analysis that otherwise would be more complex and time consuming.
This handbook takes as its reference ImageJ, open source software developed by NIH (http://imagej.nih.gov/ij/download.html).
ImageJ allows to perform operations in parallel and supports the so-called “stacks”, that is series of images that share the same window.
To open a stack of images with ImageJ click File ⊳ Import ⊳ Image Sequence and select the first image in the folder, then follow the instructions and select the desired set of images.
MicroCT data analysis with ImageJ
The steps suggested are the following:
• Check that the sample did not move during the measurements. To do this you need to compare multiple images of the same measure and make sure that the sample edges between successive images are similar and that the images do not contain artifacts that substantially alter the shape of the object studied.
• Delete and remove from your stack images without the sample, or rather those in which there is only the background.
• In the case of multiple scan along vertical axe be sure that during microCT experiments motor rotation was always set correctly. In fact, if between a first measurement and a second one the motor was not brought back to its initial position, the second image stack will be rotate by 180° in respect to the first stack.
In this case use the ImageJ tool Image ⊳ Transform ⊳ Rotate … to turn the stack.
It is convenient to reduce the image background by resizing with the Crop tool. First you have to locate a Region of Interest using the rectangular selection tool on the first stack, this selection is applied automatically to all images in the stack.
To make the image crop you must position on the first stack and select: Image ⊳ Crop and select the option to apply crop to the whole stack.
To preserve the original images new resized images must be saved in a working folder (File ⊳ Save As ⊳ Image Sequence).
If you collected different portion of the same sample, the crop has to be applied to all of the stacks related to the same sample. To do this, you must select the first stack where was positioned the ROI and select Edit ⊳ Copy, select the second stack and select Edit ⊳ Paste and Edit ⊳ Undo, this procedure must be performed on all of the same sample stacks.
Increase the image contrast
Quantitative analyses depend very much on contrast and how the edges are detectable. Images can be improved in this respect by increasing the contrast through Process ⊳ Enhance Contrast
Note that the value of “Saturated pixels” is set to 0.4%, then it can be useful to change it as explained in the next paragraphs.
Figure on the left shows an image of tooth before the contrast enhancement, on the right after the contrast enhancement. You may notice that in the second image the distinction between the phases of the tooth (background, dentin and enamel) is more pronounced.
Image conversion to 8 bits
Select the command Image ⊳ Type ⊳ 8-bit. Save 8 bits images to reduce disk space and reduce time needed to data analysis and visualization. In 8 bit format you can loose some information about the differences between very similar tissues, for example if you study regenerated bone, but in the study of tooth it is very convenient format.
Select the tool Analyze ⊳ Histogram, including all images to have quantitative feeling with your data.
The histogram contains Gaussian-like curves, on the x-axis are reported the pixel gray values which can vary between 0 and 255 (8 bit = 256 bin, being 1 byte per pixel): in the images above the value 0 indicates a black pixel, the value 255 corresponds to white pixels, intermediate values represent different level of gray. It is possible to have images where this encoding is reversed 0 white 255 black, depending on the TAG of the analyzed image which can be MIN-IS-BLACK or MIN-IS-WHITE (the image TAGS you can access with the tool Image ⊳ Show Info). On the y-axis is reported the number of pixels corresponding to a certain tone of gray (intensity). Each peak corresponds to a different phase of the sample. To know the position of the peaks relating to the different phases of the sample you must move with the cursor on the top of the curve and next to the notice “value” it will appear the relative value. In this case, for example, the first peak corresponds to the empty space and has a value of 64, the second peak corresponds to dentin and has a value of 151 and the third peak corresponds to the enamel and has a value of 212.
The figure shows the comparison between two histograms: to the left the histogram obtained before the contrast enhancement, to the right it is shown the histogram after contrast enhancement. You may notice that the contrast enhancement increases the distance between the peaks corresponding to different material phases embedded in the sample.
At this point it is appropriate to create an Excel table where, for each stack, or rather. for each measurement by microCT, to mark the peak values of the different phases.
Histogram data can be saved and later plotted with other software such as Excel or Gnuplot; to do this you must click the button below the histogram List ⊳ File ⊳ Save As.
Save histograms can be useful for quantitative analysis and the choice of thresholds to distinguish between the different phases.
Correct Bias on the histogram value (very expert user)
Comparing data in tables related to the different stacks of the same sample you will notice that the peaks corresponding to the different phases are not aligned, this can be explained by the fact that the intensity of the X-ray beam is not constant. To make quantitative comparisons on local mass density in different samples or in the same sample following a physical-chemical or biological treatment may be useful to proceed with a “manual alignment of the peaks” by following the steps below.
Repeat the contrast enhancement at different values of saturated pixels and 8 bit conversion so that in the histograms the first peak (corresponding to the empty space) has a similar value.
Create image difference
In an experiment by Synchrotron microCT often happens that due to the limited Field of View of the detector the sample has to be acquired through two or more successive scans shifting the sample along the z-axis; therefore, after processing the images as described above, the various stacks related to the same sample must be connected. In particular between one section and the next of the same sample can be present several overlapping images. It is therefore necessary to identify those overlapping images and remove them from one of the stacks
Open two images of successive stacks you believe coincident as those that follow.
Subtract the two images through the command Process ⊳ Image Calculator.
In the graphs below are reported the pixel value of the two images along a straight line, to obtain the graph use the tool Analyze ⊳ Plot Profile
If the subtraction is performed on two corresponding images the plot of pixels intensity along the line should be contain only noise. The presence of different peaks in the graph on the right side indicates that the two original pictures contain different information.
Files obtained from the vertical overlap of the stack are the starting files that can be used with the applications VDL. The software allow to import the whole image stack for the analyzed sample or more than one stack by automatically rename of the file of the consecutive stacks. This tool creates a new sequence of images representing the whole object and gives to the images a new progressive name.
For the upload procedure you can upload the data in the user area as separate images or in a single zip file. Zip file has to be preferred to save time and disk space.