Fluorescence Detection Technologies
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Authored by: Paul Taylor, Boehringer Ingleheim Inc.
Detection modalities included in this category are fluorescence intensity (FLINT), fluorescence polarization (FP), time resolved fluorescence (e.g. DELFIA), fluorescence resonance energy transfer (FRET, includes VIPR® technology), time resolved FRET (including LANCE Ultra™ and HTRF), FLIPR® ion flux and voltage, fluorescence lifetime (FLT), HCS CCD imaging and laser scanning and fluorescence correlation spectroscopy (FCS). 
Fluorescence Intensity (FLINT)
Fluorescence Intensity (FLINT) is calculated from the following formula:
Where IO is the excitation intensity, ε is the molar absorptivity, φ is the quantum yield, C is the fluorophore concentration and l is the path length. An example application of this detection mode is a fluorescent protease substrate that is peptide-linked to a fluorescence quenching molecule. Upon cleavage of the peptide bond by the protease, the quencher is cleaved, leaving the fluorescently-tagged product to freely emit.
|Image source: AnaSpec Inc.|
Fluorometric Imaging Plate Reader (FLIPR®)
Another application of FLINT is FLIPR® (Fluorometric Imaging Plate Reader). This technology has provided biologists with a way to detect the activation of G-protein coupled receptors (GPCR’s) by coupling them to Gq proteins which stimulate an intracellular calcium response upon binding. The stimulation of intracellular calcium is measured with calcium-sensitive dyes (e.g. Fluo-3, Fluo-4, Calcium 3 and Calcium 4) which are excited with a laser and detected with a cooled CCD imager. The instrument is capable of reading at intervals of less than a second and this allows monitoring of the signaling cascade which results in release of calcium from intracellular stores. Being that the responses are fast and transient, the detector operates in close conjunction with an integrated liquid handler which adds the final step to assays that have been configured to identify agonists, antagonists or allosteric modulators. The detection format also has been used to investigate ion channel targets using membrane permeable fluorescent dyes such as DiBAC4  for measuring changes in membrane potential.
|Image Source: Molecular Devices Inc.|
Fluorescence Polarization (FP)
Fluorescence Polarization (FP) uses the tumbling motion of molecules to qualify binding events. The primary concept is that if a small fluorescently-labeled molecule (such as a peptide) binds to a much larger molecule (such as a protein receptor), the tumbling speed of the fluorescent tag will significantly diminish. The tumbling speed is determined by measuring the changes in fluorescence intensity as the emission fluorescence passes through two polarized light paths: one parallel (III) to the excitation light and the other perpendicular (I⊥) to it. The polarized signal is then calculated using the following formula:
Because the fluorescence lifetime of commonly used fluorophores is <10 ns, the typical molecular mass range of FP reagents is 5-40 kDa.
|Image Source: Hybrigenics Services|
Fluorescence Correlation Spectroscopy (FCS)
Fluorescence Correlation Spectroscopy (FCS) is based on diffusion-dependent fluorescent intensity fluctuations of individual molecules as they pass through a confocal volume of ∼1 femtoliter. Using autocorrelation techniques, information is determined for diffusion times and the brightness of each molecule. As the speed of tumbling changes upon a binding event in FP experiments, so does the diffusion time in FCS experiments.
|Image Source: Stowers Institute for Medical Research|
Fluorescence Resonance Energy Transfer (FRET)
Fluorescence Resonance Energy Transfer (FRET) incorporates a non-radiative energy transfer between a fluorescent donor and acceptor. For the transfer to occur, an overlap must exist between the absorption spectrum of the acceptor and the emission spectrum of the donor. Additionally, the proximity range of the pair needs to be 1-8 nm to allow adequate energy transfer. Common FRET pairs include fluorescein/rhodamine (in vitro) or cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) (in vivo). For HTS applications, a combination of the fluorescence intensity ratio and donor lifetime variations has been used to avoid compound interference.
|Image Source: University of California San Diego|
Dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)
Dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) uses a lanthanide chelate with a long fluorescent lifetime (>100 μs) to avoid background interference from assay components such as buffers, media, reagents or compounds. The chelate protects the lanthanide ion from potential quenching and also acts as an antenna in transferring excitation energy to the lanthanide. The label absorbs light in the UV range from a nitrogen laser or flash lamp and, depending on the lanthanide used, emits fluorescence between 500 and 700 nm. While the wash steps included with this detection format can be cumbersome in an automated environment, the benefit of high detection sensitivity is commonly a key reason contributing to its selection.
The majority of commercial DELFIA kits are based on non-competitive "sandwich type" assays. The design of such assays is as illustrated for DELFIA hTSH (thyroid stimulating hormone), below.
|Image Source: PerkinElmer Life and Analytical Sciences|
Homogenous Time Resolved Fluorescence (HTRF)
Homogenous Time Resolved Fluorescence (HTRF) uses a similar molecular process as FRET but uses a lanthanide cryptate (versus a chelate) with a long fluorescent lifetime as the donor. The cryptate provides similar optical properties as the chelate in DELFIA and has been reported to have higher stability at lower pH's and less of a propensity to exchange with ions in solution. Europium cryptates are excited in the UV range by either a xenon flash lamp or nitrogen laser and emit in the 550-710 nm range with fluorescent lifetimes of 100-1,000 μs. Most commonly, europium cryptates are paired together with a cross-linked allophycocyanin (XL665). By using the long fluorescent lifetime property of the lanthanide cryptate, fluorescence interference from compounds or any unbound XL665 is effectively eliminated.
|Image Source: Cisbio-US, Inc.|
LANCE Ultra™ reagents include a series of europium chelate-labeled anti-phospho-substrate antibodies and several kinase peptide substrates directly labeled with the low molecular weight and red-shifted ULight™ dye. The direct labeling of the peptide substrate in combination with the use of the ULight™ dye provides less steric hindrance than earlier formats which utilized biotin and streptavidin as a component of capture and detection.
Fluorescence Lifetime Analysis (FLA)
Fluorescence Lifetime Analysis (FLA) gives a highly sensitive readout as an intrinsic molecular property of the fluorophore and its environment. The technique is mostly unaffected by inner filter effects, quenching or variations in the fluorophore concentration. Applications typically include binding assays where bound and unbound states of a fluorophore result in fluorescence lifetime variations between 0.5 and 20 ns.
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