Principles of Fluorescence Polarization (FP)

What is Fluorescence Polarization (FP)? — The concept of molecular movement and rotation is the basis of fluorescence polarization. By using a fluorescent dye to label a small molecule, its binding to another molecule of equal or greater size can be monitored through its speed of rotation.

Fluorescence polarization was first described in 1926 by Perrin. Weber greatly expanded the theory and developed the first instrumentation for the measurement of FP. Dandliker and co-workers expanded FP into biological systems, such as antigen-antibody reactions and hormone-receptor interactions. Jolley and co-workers developed FP into a commercial system for the monitoring of therapeutic drug levels and the detection of drugs of abuse in human body fluids. (see references to these papers in the reference library)

If we shine vertically polarized light on these molecules, the molecules with their excitation dipoles arranged perpendicular to the plane of polarized light do not absorb light and hence do not emit light. The molecules at other orientations will absorb light to varying degrees, depending on how closely they are aligned with respect to the vertical plane. The emitted light will now have a horizontal component and its resultant polarization ("limiting polarization," P₀) will be 500 mP. A polarization of greater than 500 mP indicates that there is scattered light in the system, or the instrument is incorrectly calibrated, since it is impossible to get a polarization of greater than the limiting polarization.

In the real world, we have a random array of molecules which are free to rotate. In this case, the polarization is between 0 and 500 mP and is dependent on how far the molecule has rotated during the fluorescence lifetime of the excited state. The smaller the molecule, the faster it rotates, and so the lower the FP will be.

The rate of rotation of a molecule is described by the Stokes equation:

ρ = (3ηV) / (RT)         (equation 2)

where ρ is the rotational relaxation (correlation) time (the time required to rotate through an angle whose cosine is 1/e, or approximately 68.5^o), η is the viscosity of the medium, V is the molecular volume of the molecule, R is the gas constant, and T is the temperature in degrees Kelvin. From (equations 2 and 3) we can see that the higher the molecular weight of a molecule, the higher the rotational relaxation time will be.

V=vM            (equation 3)

where M is the molecular weight of the molecule in Daltons and v is its partial specific volume (cm³ g^-1).
The Perrin equation (equation 4), which was first described in 1926, describes the relationship between the observed FP, the limiting polarization, the fluorescence lifetime of the fluorophore (τ), and its rotational relaxation time.

((1/P) - (1 / 3)) = ((1 / P₀) - (1 / 3)) x ((1 + (3τ / P))     (equation 4)

The smaller the fluorescence lifetime, the higher the FP will be. Conversely, the smaller the rotational relaxation time, the smaller the FP will be. Combining the Stokes equation and the Perrin equation, and substituting M for V and rearranging (Equation 5), we get the relationship between the molecular weight of a molecule and its FP (1 / P is proportional to 1 / M).

(1 / P) = (1 / P₀) + ((1 / P₀) - (1 / 3)) x (RT / vM) x (τ / η)     (Equation 5)

From equation 5 we can see that P equals P₀ at infinitely high molecular weight, infinitely high viscosity, and infinitely short lifetime. In fact, P₀ can be determined by measuring FP at various viscosities, plotting P against 1/η, and determining the intercept on the ordinate.

Why Use Fluorescence Polarization (FP)? — FP is a homogeneous technology and reactions are very rapid, taking seconds to minutes to reach equilibrium. The reagents are stable. Large batches may be prepared. This results in high reproducibility. Because of these properties, FP has proven to be highly automatable, often performed with a single incubation with a single, premixed, tracer-receptor reagent. The fact that there are no washing steps increases the precision and speed over heterogeneous technologies and dramatically reduces waste.

Other homogeneous technologies based on fluorescence intensity have been developed. These include energy transfer, quenching, and enhancement assays. FP offers several advantages over these. The assays are usually easier to construct, since the tracers do not have to respond to binding by intensity changes. In addition, only one tracer is required and crude receptor preparations may be utilized. Furthermore, since FP is independent of intensity, it is relatively immune to the inner filter effect and so works in colored solutions and cloudy suspensions. FP offers several advantages in the area of instrumentation. Because FP is a fundamental property of the molecule, and the reagents are stable, little or no standardization is required. FP is relatively insensitive to instrument changes such as drift, gain settings, lamp changes, etc. In addition, fluorescence intensity is obtained in addition to polarization, if required.

FP Instrumentation

FP is instrument dependent, the quality of the results is directly linked to the quality of the instrument.

Click here to return to the top of the page.