ABSTRACT
Fluorescence is a highly bene›cial modality for biomedical imaging as it imparts versatile contrast of cellular and subcellular function and structure. As a result, it has been overwhelmingly utilized in the biomedical laboratory for performing in vitro assays, immunohistochemistry, or the super›cial visualization of cells in vivo. ™e compelling advantages of ¦uorescence have more recently driven the development of powerful classes of ¦uorescent tags that can stain functional and molecular processes in vivo. ™e most widely acknowledged technology is naturally the 2008 Nobel-prize-awarded ¦uorescent protein
(FP), which ožers perhaps the most versatile tool for biological imaging (Giepmans et al. 2006). FPs attain the ability to tag cellular motility and subcellular process, from gene expression and signaling pathways to protein function and interactions, merging optimally with post-genomic “-omics” investigations and interrogating biology at the systems level. Promising new developments include the introduction of truly near-infrared (NIR)-shi²ed FPs, with excitation and emission spectra in the >650 nm (Shu et al. 2009). Such performance opens exciting possibilities for whole body animal imaging as it allows highsensitivity imaging through several centimeters of tissue, due to the low photon attenuation by tissue in the 650-950 nm range, that is, the NIR spectral region. In parallel, a plethora of extrinsically administered probes are being developed, also operating in the NIR region (Tsien 2005, Weissleder and Pittet 2008). Fluorescent probes are agents that can probe tissue constituents and their function by staining in vivo certain classes of cells, receptors, proteases, and other moieties of cellular or subcellular activity. During the last decade, a large number of experimental and commercially available ¦uorescent agents and probes are increasingly being ožered, from nonspeci›c ¦uorescent dyes and various FPs to targeted or activatable photoproteins and ¦uorogenic-substrate-sensitive ¦uorochromes to enable a highly potent ›eld for biological imaging. So far, these contrast mechanisms were proven eŸcient in a number of small-animal
22.1 Introduction ............................................................................................................................ 443 22.2 Reporter Technologies ........................................................................................................... 444
22.5 Discussion ................................................................................................................................ 454 Acknowledgments .............................................................................................................................. 455 Glossary ............................................................................................................................................... 455 References ............................................................................................................................................ 456
applications but many of these agents attain strong potential for clinical translation as well. In addition, voltage sensitive dyes, ¦uorescence resonance energy transfer approaches, and lifetime measurements further allow the sensing of ions, protein-protein interactions, or the ežects of the biochemical environment on the ¦uorochrome (Homma et al. 2009). Using ¦uorescence, therefore, previously invisible processes associated with tissue and disease growth and treatment can be sensed and visualized in real time and longitudinally. Naturally, ¦uorescence is increasingly used in basic biological discovery, drug discovery, and even considered for clinical studies of cancer, in¦ammation, neurodegenerative disease, cardiovascular disease, to name a few examples.
