Fluorescence polarization/anisotropy in diagnostics and imaging.

Fluorescence polarization was first applied in biochemistry almost 6 decades ago, when Gregorio Weber described his studies on bovine serum albumin and ovalbumin conjugated with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride)1,2 (Figure 1). (For an overview of Gregorio Weber's wide-ranging contributions to fluorescence see3). Polarization methods became increasingly popular during the decades following Weber's work. During the past few decades, however, the increase in the number and diversity of fluorescence polarization studies has been astonishing and the method is now extremely wide-spread in the clinical and biomedical fields. The virtual explosion of polarization studies, which began during the mid-1980's, was due to several factors, including the availability of commercial instruments equipped with polarizers, the commercial availability of a great many fluorescence probes (largely due to the company Molecular Probes – now part of Invitrogen) and, in the clinical chemistry area, the introduction of the TDx instrument (and associated reagents) by Abbott Laboratories. The reasons for the popularity of fluorescence polarization in clinical and high-throughput assays are manifold. Firstly, polarization assays are homogeneous, i.e., there is no necessity for separation of free and bound ligand (these type of assays are often referred to as “mix and measure” assays). Secondly, and one of the original motivations for the development of fluorescence-based assays, there is no need for radioisotopes. Thirdly, polarization assays are reproducible and facilely automated. In this review we shall briefly trace the history of the technique and discuss in detail both theoretical and practical aspects. We shall also present copious examples from the literature, which illustrate the scope of the method in areas such as ligand binding, immunoassays, high-throughput screening, and live cell imaging and hint at future directions and applications.