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Purpose: 2-[F]Fluoro-2-deoxy- D-glucose (F-FDG) is a widely used PET radiotracer for the in vivo diagnosis of several diseases such as tumours. The positrons emitted by F-FDG, travelling into tissues faster than the speed of light in the same medium, are responsible for Cerenkov radiation (CR) emission which is prevalently in the visible range. The purpose of this study is to show that CR escaping from tumour tissues of small living animals injected with F-FDG can be detected with optical imaging (OI) techniques using a commercial optical instrument equipped with charge-coupled detectors (CCD). Methods: The theory behind the Cerenkov light emission and the source depth measurements using CR is first presented. Mice injected with F-FDG or saline solution underwent dynamic OI acquisition and a comparison between images was performed. Multispectral analysis of the radiation was used to estimate the depth of the source of Cerenkov light. Small animal PET images were also acquired in order to compare the F-FDG bio-distribution measured using OI and PET scanner. Results: Cerenkov in vivo whole-body images of tumour-bearing mice and the measurements of the emission spectrum (560-660 nm range) are presented. Brain, kidneys and tumour were identified as a source of visible light in the animal body: the tissue time-activity curves reflected the physiological accumulation of F-FDG in these organs. The identification is confirmed by the comparison between CR and F-FDG images. Conclusion: These results will allow the use of conventional OI devices for the in vivo study of glucose metabolism in cancer and the assessment, for example, of anti-cancer drugs. Moreover, this demonstrates that F-FDG can be employed as it is a bimodal tracer for PET and OI techniques. [ABSTRACT FROM AUTHOR]