[en] The aim of this study was to characterize the in vivo volumetric distribution of three folate based biosensors by different imaging modalities (X-ray, fluorescence, Cerenkov luminescence and radioisotopic imaging) through the development of a tri dimensional (3D) image reconstruction algorithm. The preclinical and multimodal Xtreme imaging system, with a Multimodal Animal Rotation System (Mars), was used to acquire bidimensional (2D) images, which were processed to obtain the 3D reconstruction. Images of mice at different times (biosensor distribution) were simultaneously obtained from the four imaging modalities. The filtered backprojection and inverse Radon transformation were used as main image-processing techniques. In the first instance, the algorithm developed in Mat lab was able to reconstruct in the 3D form the skeleton of the mice under study. Subsequently, the algorithm was able to get the volumetric profiles of "9"9"mTc-Folate-Bombesin (radioisotopic image), "1"7"7Lu-Folate-Bombesin (Cerenkov image), and FolateRSense 680 (fluorescence image) in the tumors and kidneys of the mice. No significant differences were detected between the volumetric quantifications using the standard measurement techniques and the quantifications obtained with the proposal made in this study, nor between the volumetric uptakes in the structures of interest. With the structures reconstructed in the 3D form, the fusion of anatomical (as the skeleton) and functional structures derived from the images of the biosensors uptake was achieved The imaging 3D reconstruction algorithm can be easily extrapolated to different 2D acquisition-type images. This characteristic flexibility of the algorithm developed in this study is an advantage in comparison to similar reconstruction methods. (Author)