The most basic fluorescence technique involves exciting a sample with light at one frequency and then measuring the output at a different frequency. Several technologies can improve the sensitivity and get around some of the limitations. Confocal optics, such as LJL Biosystems' SmartOptics technology, can be used to maximize sensitivity by detecting fluorescence in a chosen region of the assay volume. For homogeneous samples in solution, such as enzymatic assays, the optimum signal is obtained near the top of the sample, where background fluorescence from the microplate is minimized. For cellular-based assays, the optimum signal is obtained from the cellular layer near the bottom of the sample, where background fluorescence from the buffer is minimized.
Optical filters are also widely used to enhance sensitivity and reduce background fluorescence.
By choosing a fluorophore with a large Stokes shift, background fluorescence can be minimized and sensitivity enhanced. Since 1980, FP immunoassays have been used extensively in the clinical laboratory to measure the concentration of drugs, both therapeutic and illicit, in biological fluids. Recently, LJL Biosystems, PanVera, and other companies have adapted the FP technique to the HTS environment. FP theory states that when a molecule is excited with plane-polarized light of the correct wavelength, it will fluoresce in the same plane after its characteristic emission lifetime, which is typically a few nanoseconds. During this time, the molecule will have tumbled randomly with respect to the original plane of excitation.