Fluorescence methods are also fast and nonradioactive. Some techniques can be used with samples that are colorized or that use cellular samples. Given all the advantages of fluorescence, many experts are now seeing a shift away from radioactive assays and toward fluorescence assays. Assay developers have demonstrated that most of the radiometric assay types have an analogous fluorescence approach. 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.