How does fluorescence polarization work?
Basic principle of fluorescence polarization. A fluorophore is excited with light that is linearly polarized by passing through an excitation polarizing filter; the polarized fluorescence is measured through an emission polarizer either parallel or perpendicular to the exciting light’s plane of polarization.
What is the difference between fluorescence polarization and anisotropy?
Fluorescence polarization and anisotropy are mathematically related and used interchangeably. Both are derived from the emission intensities of polarized and unpolarized light and represent an average of the bound/unbound states of a fluorescent molecule.
How do you measure fluorescence polarization?
Fluorescence polarization is then measured as the difference of the emission light intensity parallel (I||) and perpendicular (I⊥) to the excitation light plane normalized by the total fluorescence emission intensity.
How does fluorescence anisotropy work?
Fluorescence anisotropy is sensitive to factors that affect the rate of the rotational diffusion of the fluorophore and thus depends on the temperature, the viscosity of the solution and the apparent molecular size of the fluorophore.
What is G factor in fluorescence polarization?
The G factor (G) is used to correct for the effects of optical components like filters, polarizers, and monochromators, which can affect polarization values. Unbound tracer. Tracer bound to a larger molecule. The larger the fluorescently labeled molecule, or the molecule to which tracer is bound, the larger the mP …
What is fluorescence polarization anisotropy?
Fluorescence anisotropy or fluorescence polarization is the phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarization.
Why Fluorescence Polarization Immunoassay is important?
Fluorescence polarization immunoassay (FPA) is a homogeneous immunoassay useful for rapid and accurate detection of antibody or antigen. The principle of the assay is that a fluorescent dye (attached to an antigen or an antibody fragment) can be excited by plane-polarized light at the appropriate wavelength.
What is drug and FP?
Fluorescence polarization assays (FP assays) are used in a variety of ways. From uncovering molecules in solution, to monitoring drug levels in clinical settings, and are enabling in drug discovery.
How do you calculate G factor in anisotropy?
Since there is no difference between the number of photons coming towards the HH and HV channels from the sample, G is calculated as the ratio between the measured total intensities (counts) in each channel: Note that some software use the opposite definition of the G-factor (i.e. G = HH/HV).
What causes Stokes shift?
The Stokes shift is primarily the result of two phenomena: vibrational relaxation or dissipation and solvent reorganization. A fluorophore is a dipole, surrounded by solvent molecules. When a fluorophore enters an excited state, its dipole moment changes, but surrounding solvent molecules cannot adjust so quickly.
What causes fluorescence quenching?
Fluorescence quenching is a physicochemical process that lowers the intensity of emitted light from fluorescent molecules. When a molecule absorbs light, electrons in its constituent atoms become excited and are promoted to a higher energy level.