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The interest in optical molecular imaging of small animals in vivo has been steadily increased in the last two decades as it is being adopted by not only academic laboratories but also the biotechnical and pharmaceutical industries. In this Spotlight paper, the elements for in vivo optical molecular imaging are briefly reviewed, including contrast agents, i.e., various fluorescent reporters, and the most commonly used technologies to detect the reporters. The challenges particularly for in vivo fluorescence imaging are discussed and solutions to overcome the said-challenges are presented. An advanced imaging technique, in vivo fluorescence lifetime imaging, is introduced together with a few application examples. Taking advantage of the long fluorescence lifetime of quantum dots, a method to achieve background-free in vivo fluorescence small animal imaging is demonstrated.