Optical projection tomography (OPT) is a novel three-dimensional imaging technique, which provides an approach to recreating the three-dimensional images of biological specimens ranging from millimeters to centimeters. In the current OPT setup, the specimen was immersed in the index-matching fluid. In this case, the specimen could not survive or the survival time was too short for longitudinal imaging. In this paper, we first designed a new type of sample fixation method for in vivo OPT imaging. The specimen was embedded into a transparent gel in a petri dish, and the dish was affixed to the rotational stage of our homemade OPT system. As the specimen does not need to be immersed in the index-matching fluid, this method can reduce the damage to the specimen and it is more conducive to longitudinal observation for in vivo OPT. However, this fixation method induces a problem of insufficient measurements. The angles parallel to or nearly parallel to the surface of the dish cannot be acquired. To address this problem, we, then, used a limited-angle reconstruction framework for the novel sample fixation-based OPT, which combines the algebraic reconstruction technique (ART) algorithm with prior information to solve the inverse reconstruction problem. The feasibility and effectiveness of the proposed sample fixation method and the limited-angle reconstruction framework were verified by the simulations and experiments. INDEX TERMS Optical projection tomography, in vivo imaging, limited-angle reconstruction, sample fixation. XUELI CHEN received the B.S. degree in biomedical engineering and the Ph.D. degree in pattern recognition and intelligent system from Xidian University, Xi'an, China, in 2007 and 2012, respectively, where he is currently a Professor with the School of Life Science and Technology. From 2015 to 2017, he was a Postdoctoral Researcher with Purdue University. His research interest includes multi-scale and multi-modality biophotonics imaging and its applications. He has authored or coauthored more than 60 peer-reviewed journal papers on these topics. He is currently a member of OSA and SPIE. SHOUPING ZHU received the B.S. degree in biomedical engineering and the M.S. degree in signal and information processing from Xidian University, Xi'an, China, in 2004 and 2007, respectively, and the Ph.D. degree in pattern recognition and intelligent systems from the Chinese Academy of Sciences, Beijing, China, in 2010. He is currently a Professor with the School of Life Science and Technology, Xidian University. His research interests include multi-modality medical imaging systems, micro-CT imaging, PET imaging, optical imaging, and tomography reconstruction with GPU.