• Fluorescence
• Absorbance
• Luminescence
• Time-Resolved Fluorescence (TRF)
• HTRF®
• Fluorescence Polarisation (FP)
• BRET
• AlphaScreen®

Both filter- and monochromator-based microplate readers are available. Find out more about the detection modes below:

UV/VIS Absorbance or Optical Density is used in countless classical colorimetric methods, ELISA, nucleic acid and protein quantification, and many others. The basic principle of absorbance measurements is described by Lambert-Beer's law.

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Methods based on fluorescence offer better sensitivity, specificity and dynamic range than similar methods based on absorbance. Fluorescence is also involved in methods such as FRET and Fluorescence Polarisation (see below). Fluorescence measurements are performed very often using multimode microplate readers.

Luminescence is the result of a chemical or biochemical reaction. Thus, no excitation energy is required. Luminescence is most frequently used in methods involving reporter genes, ATP measurement and CLIA (Chemiluminescence Immunoassays). Other luminescence applications are caspase or kinase assays and ROS. Luminescence is usually measured using either microplate luminometers or tube luminometers.

BRET(Bioluminescence Resonance Energy Transfer) is based on the fact that the energy derived from a luciferase reaction can be used to excite a fluorescent protein if the latter is in close proximity to the luciferase enzyme, and is thus, an excellent tool to monitor macromolecular interactions. As many dedicated luminometers lack filters, multimode microplate readers are usually the instrument of choice for BRET measurements.

FRET (Fluorescence Resonance Energy Transfer) is a dual dye fluorescence detection assay. It requires a donor and an acceptor dye being in close proximity (10-100 Å). As a result of excitation, the donor fluorophore can transfer part of its energy to the acceptor fluorophore, e.g. YFP and starts emitting light without being directly excited. FRET is most frequently used to visualize molecular interactions of proteins and nucleic acids.

Fluorescence Polarisation (FP) is ideal to measure the binding of a small molecule to a much larger one. It is based on a fluorophore being excited by polarized light. Proteins and other large molecules in solution rotate slowly due to their size and emit polarized light when getting excited by polarized light. Small molecules on the other hand rotate faster and emit depolarized light. Thus, high levels of polarization indicate the presence of a larger molecule.

AlphaScreen® relies on hydrogel coated donor and acceptor beads providing functional groups for conjugation to biomolecules. When excited with a laser at 680 nm the donor bead produces singlet oxygen which transfers energy to the donor resulting in light emission between 520 and 620 nm. The amount of light produced is directly proportional to the amount of bound donor-acceptor beads. AlphaScreen® is a versatile method used to evaluate interactions between a binder (or a collection of binders) and a target (or a collection of targets). Only microplate readers equipped with a laser are suitable for AlphaScreen® measurements.

Time-Resolved Fluorescence (TRF) implies the use of fluorophores with a distinctive fluorescence lifetime to avoid the interference caused by molecules with a very different fluorescence lifetime or other factors (most importantly, excitation light). It uses long emission fluorophores (lanthanides) such as Europium or Terbium. TRF is used to elucidate molecular interactions, inter- or intramolecular distances, changes in the microenvironment, resolution of molecular mixtures, and more. A flash lamp or laser are the light sources of choice in any microplate reader to be used for Time-Resolved fluorescence measurements.

TR-FRET (Time-resolved fluorescence energy transfer) is a variant of FRET using long emission fluorophores (lanthanides) as donors. TR-FRET represents a powerful tool for drug discovery researchers. The resulting assay provide an increase in flexibility, reliability and sensitivity in addition to higher throughput and fewer false positive/false negative results. TR-FRET can be applied to detect the association of two molecules, for example to analyze receptor-ligand or protein-protein interactions.