High Content Screening


Object-Based Fluorescence Polarization or Anisotropy

Fluorescence continues to be a powerful tool for life science research due to its sensitivity, selectivity, and general usability. Typically, the presence and amount of a fluorescently-tagged analyte is correlated to the intensity of the fluorescence measured. This can be done in many formats including bulk biochemical assays, spot arrays, or bead and cell-based assays. Multiple emission colors can be used to multiplex analytes within the same assay.

Laser Scanning Cytometry

The majority of fluorescence-based assays are performed in bulk solution as relatively well-controlled biochemical assays. Laser scanning offers a two-dimensional image of fluorescence emitting from objects (spot, beads, cells, etc.) on the surface of slides or microplate well bottoms. Traditionally this has been a method of choice for reading arrays for nucleic acids, proteins, and adherent cells. In laser scanning cytometry, a small micron sized laser spot is rastered along a surface on which the array elements, beads, or cells are located. At every position that the spot covers, the emission intensity is recorded and a two-dimensional intensity map can be reconstructed. The reconstructed map is essentially an image of the surface containing the fluorescent objects.

Fluorescence Polarization

The use of fluorescence polarization (FP) builds on these techniques by using a fluorophore's properties to indicate binding state and microenvironment. Polarization is a measurement of a molecule's rotation from its excitation until its emission. Polarization (P) is defined as:

Anisotropy FP equation

Where I|| is the emission intensity parallel to the excitation plane and I is the intensity perpendicular to the excitation plane.

Anisotropy FP diagram

If the emission is completely maintained in the parallel direction then P=+1. If the emitted light is totally polarized in the perpendicular direction then P=-1. In the actual measurement of polarization there is usually an angle between the excitation dipole and the emission dipole within a molecule. For example, even a fixed, non-rotating fluorophore, will have some "intrinsic" depolarization due to this angle. See comparison of Laser Scanning Cytometer to Analyst for bulk solution biochemical fluorescence polarization assays like IMAP (link to app note xx and FP poster ).


Another term representing the degree of polarized emission is anisotropy (r) which is defined as:

Anisotropy equation

The difference in the way polarization and anisotropy is defined is the presence of a second perpendicular intensity term in the denominator. The definition of anisotropy takes into account the second possible perpendicular emission plane, which is oriented along the axis of propagation. Anisotropy is a more accurate representation of the physical phenomena because it takes into consideration the contribution of all degrees of rotational freedom and reflects orientations of constrained molecules that can be detected through two-dimensional mapping of polarized fluorescence emission.

Relating Fluorescence Polarization and Anisotropy

Given the definition of polarization and anisotropy, one can show a correlation between polarization and anisotropy values. 









The information content in the polarization function and the anisotropy function is identical and the use of one term or the other is dictated by practical considerations.

Rotational Mobility and Lifetime

Mobility and lifetime are the two factors that determine anisotropy. No matter how fast a molecule is rotating, if its lifetime is short enough, there will be a high anisotropy, i.e., very little perpendicular emission. Likewise, if a large, slowly rotating molecule has a very long lifetime, there will also be a high anisotropy. Hence, both size (rotational mobility) and fluorescence lifetime can influence anisotropy. A molecule rigidly bound to a cell or bead surface or cellular organelle is essentially an infinitely large molecule, and binding assays based on anisotropy can offer a sensitive, powerful approach to converting these interactions into screening assays.

Click here to learn more about the ImageXpress Velos Laser Scanning Cytometer.