For all cameras
When taking the test photographs for establishing the proper autofocus adjustment value, it is critical that as many variables as possible be eliminated. The physical position of the camera is one of those variables. So when de-focusing as well as when releasing the shutter we want to ensure that the camera to be moved as little as possible.
To that end, it is important to use the shutter release button to also engage the autofocus system. If we were to use the autofocus enable button located on the back, we have an additional chance of physically moving the camera, which would result in slight shot to shot variation in terms of the camera position. By using the top located shutter release button, both for taking the picture and for enabling autofocus, we have one less button to press. Ideally, and highly recommended is to actually use the self timer or cable release, to further reduce any movement from occurring.
Note: Within the FocusTune image processing engine, all of the test photographs are actually aligned to one another within 1pixel accuracy, so that camera movement is taken out of the equation. But the less FocusTune has to adjust the images, the more accurate the test results will be.
For Canon cameras
When using the rear autofocus enable button on Canon cameras, important metadata critical to the FocusTune process is improperly written to the image file. We are not sure if this is a bug or a design decision, but in either case there is no upside and it will prevent FocusTune from properly analyzing the image files. So in addition to the reasons stated in the first section above, when using Canon cameras, the rear autofocus enable button simply cannot be used in conjunction with FocusTune.