Brain normalization is the rigid rotation and/or deformation of a 3D brain scan so that it can match a template brain. This is a necessary step for the analysis of brain magnetic resonance data (whether it is morphological or functional) as well as of brain PET data. It allows introducing a set of spatial coordinates such that each triplet (x,y,z) identifies the same anatomical region both in the brain we are studying and in the template (Mandal P.K. et al. 2012). So, for instance, once the normalization has been performed on an fMRI of the brain of a patient and on a set of fMRIs from a suited control group, we can compare the BOLD activation of each anatomical region of the patient’s brain with the activation of the corresponding anatomical region of the control group.

Mathematical notes

This part can be skipped, it is not necessary to read these passages for the understanding of the following paragraphs. If we assume that P is a point of the brain before normalization and we call S(P) its position after the normalization, we can consider the vectorial function:

formula 1

which gives the new position of each point of the brain, after normalization. If P_0 is a point of the brain before the normalization, then we can write:

formula 2

and remembering the expression of the differential of a vectorial function, we have

formula 3

With a few passages we can write:

formula 4

From the above formula, we realize that in order to define the configuration of the brain after normalization, we have to define, for each point P, a set of 12 parameters. Of these parameters, 6 describe the rigid movement and can be considered the same for each point. The other 6 (the coefficients of the matrix) are those that describe the change of shape and size and, in general, they are different for each point. The job of SPM is to calculate these parameters.

Brain atlases

There are several templates (also called brain atlases) that have been developed across the years. The very first one was published in 1957 (Talairach J. et al. 1957). The same researcher then participated in building one of the most used brain atlas ever, based on a single female brain, published in 1988 (Talairach J. et Tournoux P. 1988). Another widely used template is the so-called MNI-152. It was built adapting the 3D MRI brain scans of 152 healthy individuals to the Talairach and Tournoux template. The adaptation was achieved using both a rigid roto-translation and a deformation (Maintz J.B. et Viergever M.A. 1988). The second step is required for overcoming the problem of differences in brain shape and dimension that we encounter within the human population.


Available brain atlases have some limitations. One of them being the fact that despite diseased brains are the most widely studied, they are also the most difficult to register to a template built from healthy individuals, because of usually marked differences in shape and/or size. This is true for instance for brains of patients with Alzheimer’s disease (Mandal P.K. et al. 2012). Another important limitation is that registration algorithms perform poorly for the brainstem (particularly for pons and medulla) (Napadow V. et al. 2006). This might have represented a problem for the study of diseases where a possible involvement of the brainstem is suspected, like for instance ME/CFS (VanElzakker M. et al. 2019).


The SPM software package is one of the most widely used instruments for the analysis of functional brain imaging data (web page). It is freely available for download, but it requires that you have a MatLab copy in your computer. Those who don’t have a MatLab license can request and install a standalone version of SPM12 by following the instructions of this page.

Importing a DICOM file

Once you have installed SPM12 in your computer, the first step in order to register a brain is to convert the format the series of images are written in, to a format that SPM12 can read. MRI images are usually in .dcm format (DICOM) while SPM12 reads .nii files. In order to do that, click DICOM import (figure below, on the left, red ellipse), then click on DICOM files (on the right, red ellipse), then select your .dcm file and click DONE (below, red ellipse). If you then click DISPLAY (blue ellipse, left) you will see your MRI scan in another window (see next paragraph). A video tutorial on these operations is available here.

DICOM import

Setting the origin

To start the normalization process, it is highly recommended to set manually the origin of the coordinates. If this is done properly, the registration will not only take less time but, even more importantly, the chances of a successful normalization will increase. The origin is set at the level of the anterior commissure (figure below). To find this anatomical structure, you can follow this video. Once you have put the cross on the right place in the sagittal, coronal and axial windows, just click SET ORIGIN (red ellipse) and then save your work clicking REORIENT (blue ellipse).


Normalization estimate

In this step, SPM12 calculates the set of distortions that have to be applied to the source brain to adapt it to the template in MNI space. On the main menu select NORMALIZE (ESTIMATE) (figure, on the left, red). This will open the batch editor where you are asked to load the subject you want to apply normalization to (figure, right, red). You have also a set of estimation options (blue), that we leave as they are.  Then you click the RUN button, the arrow on the top of the batch editor.


At this point, your PC will perform a set of calculations that will require a few minutes. At the end of this process, a new .nii file will be saved in the spm12 folder. This is the set of distortions that will allow your subject’s brain to be registered to the template.

Normalization writing

Now click on NORMALIZE (WRITE) on the main menu. The batch editor will then ask you for the deformation field, which is the file generated in the previous step, and for the images to write, which is the scan of your subject (figure below). Select them, then press the RUN button on the batch editor. A new .nii file will be written in the spm12 folder. This is the normalized brain!

Normalization 2

In the next figure, you have the normalized brain on the left and the initial scan of the same subject on the right. As you can see, there is an overall change of shape.

Normalization 3

Anatomical areas

Now that we have normalized our brain in the MNI space, we can easily find anatomical regions within its sections. We can, for instance, load the normalized brain with MRIcron and overlay a template with Brodmann’s areas highlighted in various colours (figure below).


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