Hi Fabian,
from what I can tell, it sounds like you are trying to setup your program such that the pupil appears in the same place for every image.
In order to do this, I would begin by finding the pupil within the image. You might be able to do this by finding the concentric circles that make up the pupil and iris. Identify the pupil, and use the center point of the pupil as a reference point to establish a co-ordinate system.
Once a co-ordinate system is established, the program will use this as the basis for your future measurements. The co-ordinate system will ensure that any measurements you make will be in reference to the location of the pupil.
If you want to find more information on co-ordinate systems, I would recommend a particular example VI. The "Battery Clamp Inspection Example" ships with Labview and can be found at:
C:\Program files\National Instruments\Labview 7.1\examples\Vision\3. Applications\Battery Clamp Inspection Example.vi
To answer your second question, I would think that you could perform this using both color and grayscale images. If you use a color image, you might consider averaging the center point of the pupil for all three color planes. This might give you a more accurate center point.
Good luck, and let us know how things go!
Robert