To begin, launch the image processing software, select file, then open data and choose the previously generated micro CT scanned images in dot BMP format. When the window of the image read parameters opens, confirm the parameters and accept if correct. In the main panel, click on project view to locate a new object created with the name of the dot BMP file.
Right click on the created object and select the option ortho slice, then click create. Under properties, select the orientation plane and the slice number to see. To obtain 3D volumes of the complete head in the main panel, go to project view, right click the image object, select the interactive thresholding option, and click create.
Under properties, change the values for minimum threshold RGB and maximum threshold RGB until the part of the image corresponding to the head of the animal is selected. Then select apply. Right click on the threshold object created under the project view.
In properties select isosurface, choose a threshold to visualize the surface, then click apply. To digitize the landmarks, right click on project view, select create object, then select points and lines followed by landmarks, then click on create. Then right click the new object landmarks.
Select landmark view and click on create. Under size, modify the point size. To add landmarks in properties, in the edit mode, select add.
Digitize five equally distributed points around the curves until the next landmark at the midbrain cortex intersection, then digitize eight semi landmarks around the cortex. To save the digitized points right click on object landmarks and choose save data. To segment the brain, right click on project view, followed by create object, select images and fields, and then label field.
Click on create. Select the label field object, and press the segmentation editor option in properties. Under materials, add a new material and rename it as brain.
Under selection, choose the current slice to segment the tissue corresponding to the brain in each slice. In tools, use the magic wand and brush to select the brain tissue in each slice. Then under selection, press add.
Now return to project view, right click on label field and select generate surface, then click apply. Once the surface is obtained, apply extract surface to export it. With micro CT scanning, despite the small size of neonatal mouse brains, the anatomical structures such as the olfactory bulbs, cortex, midbrain, cerebellum, and hind brain can be distinguished.
The manual segmentation of the entire brain resulted in the 3D model reconstruction of the neonates brain.
