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Since it was first presented in 2002, optical projection tomography (OPT) has emerged as a powerful tool for the study of biomedical specimen on the mm to cm scale. In this paper, we present computational tools to further improve OPT image acquisition and tomographic reconstruction. More specifically, these methods provide: semi-automatic and precise positioning of a sample at the axis of rotation and a fast and robust algorithm for determination of postalignment values throughout the specimen as compared to existing methods. These tools are easily integrated for use with current commercial OPT scanners and should also be possible to implement in “home made” or experimental setups for OPT imaging. They generally contribute to increase acquisition speed and quality of OPT data and thereby significantly simplify and improve a number of three-dimensional and quantitative OPT based assessments.