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. Figure 1 Gregorio Weber in Hawaii – 1989. Photograph courtesy of Prof. David Jameson. 2.0 Historical Overview As mentioned above, fluorescence polarization is widely applied in diverse fields, especially in the life sciences. A rough survey using Pubmed reveals that publications on fluorescence polarization/anisotropy numbered around a dozen in the 1950's, less than 100 in the 1960s, several hundreds in the 1970s, more than two thousand in the 1980's, more than three thousand in the1990s and close to four thousand in the 2000s to date. Given this rising trend, one may be curious how it all got started. We shall thus present a brief history of fluorescence polarization. 2.1 Discovery of Polarization In 1808, Etienne-Louis Malus observed sunlight reflected from the windows of the Luxemburg Palace in Paris through an Iceland spar (Calcite) crystal that he rotated (Erasmus Bartholin had discovered the double refraction of light by Iceland spar in 1669). Malus discovered that the intensity of the reflected light varied as he rotated the crystal and coined the term “polarized” to describe this property of light. He published his findings in 1809 4. Malus also derived an expression for calculating the transmission of light as a function of the angle (θ) between two polarizers. This equation (Malus' Law) is now written as: I(θ) = I0 (cos2θ). Some years later, David Brewster studied the relationship between refractive index and angle of incidence on the polarization of the reflected light 5. He discovered that for normal glass and visible light, an incidence angle of ∼56 degrees resulted in total reflection of one plane of polarization – this angle is now known as Brewster's Angle. This discovery allowed Brewster to construct a polarizer composed of a “pile of plates” (interestingly, when one of the authors – DMJ – first joined Gregorio Weber's laboratory as a graduate student, Weber showed him the “pile of plates” polarizer that he had used in some of his initial studies). In 1828, William Nicol joined two crystals of Iceland spar, cut at an angle of 68°, using Canada balsam, which allowed a spatial separation between the orthogonally polarized components. Other important calcite polarizers developed around this time include: Glan-Foucault; Glan-Thompson; Glan-Taylor; Wollaston; and Rochon. Most modern spectrofluorimeters today use Glan-Taylor type calcite polarizers, which have an air-gap between the two calcite crystals allowing for transmission deep into the ultraviolet, and which makes them less susceptible to photodamage at higher irradiance levels. But the Henry Ford of polarizers was Edwin Herbert Land. In 1929 Edwin Land patented the sheet polarizer (the J-sheet), consisting of crystals of iodoquinine sulfate embedded in nitrocellulose film followed by alignment of the crystals by stretching, which led to dichroism. In 1937 he founded the Polaroid Company and in 1938 he invented the H-sheet which comprised polyvinyl alcohol sheets with embedded iodine. Land's invention made him quite rich and also allowed the use of inexpensive polarizers in diverse applications such as sunglasses and photography filters.