Time-resolved fluorescence energy transfer

Last updated

Time-resolved fluorescence energy transfer (TR-FRET) is the practical combination of time-resolved fluorometry (TRF) with Förster resonance energy transfer (FRET) that offers a powerful tool for drug discovery researchers. TR-FRET combines the low background aspect of TRF with the homogeneous assay format of FRET. The resulting assay provides an increase in flexibility, reliability and sensitivity in addition to higher throughput and fewer false positive/false negative results. FRET involves two fluorophores, a donor and an acceptor. [1] Excitation of the donor by an energy source (e.g. flash lamp or laser) produces an energy transfer to the acceptor if the two are within a given proximity to each other. The acceptor in turn emits light at its characteristic wavelength.


The FRET aspect of the technology is driven by several factors, including spectral overlap and the proximity of the fluorophores involved, wherein energy transfer occurs only when the distance between the donor and the acceptor is small enough. In practice, FRET systems are characterized by the Förster's radius (R0): the distance between the fluorophores at which FRET efficiency is 50%. For many FRET fluorophore pairs, R0 lies between 20 and 90 Å, depending on the acceptor used and the spatial arrangements of the fluorophores within the assay. [1] Through measurement of this energy transfer, interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer. Acceptor emission as a measure of energy transfer can be detected without needing to separate bound from unbound assay components (e.g. a filtration or wash step) resulting in reduced assay time and cost. [2]


Homogeneous, mix-and-read TR-FRET assays offer advantages over other biomolecular screening assays, such as fluorescence polarization (FP) or TRF assays. [3] In FP assays, background fluorescence due to library compounds is normally depolarized and background signal due to scattered light (e.g. precipitated compounds) is normally polarized. Depending on the assay configuration, either case can lead to a false positive or false negative result. However, because the donor species used in a TR-FRET assay has a fluorescent lifetime that is many orders of magnitude longer than background fluorescence or scattered light, emission signal resulting from energy transfer can be measured after any interfering signal has completely decayed. TR-FRET assays can also be formatted to use limiting receptor and excess tracer concentrations (unlike FP assays), which can provide further cost savings. [4] In the case of TRF assays, a wash step is required to remove unbound fluorescent reagents prior to measuring the activity signal of the assay. This increases reagent use, time to complete the assay, and limits the ability to miniaturize the system (e.g. converting from a 384-well microtiter plate to a 1536-well plate). [5] TR-FRET assays take advantage of the required proximity of the donor and acceptor species for generation of signal.

Additionally, this method is preferred by some researchers as it does not rely on radioactive materials to generate the signal to be detected. This avoids both the hazards of using the materials and the cost and logistics of storage, use, and disposal. [6]


Although TR-FRET can be accomplished with a variety of fluorophore combinations, lanthanide metals are particularly useful. Certain life science applications take advantage of the unique fluorescence properties of lanthanide ion complexes (Ln(III) chelates or cryptates). These are well-suited for this application due to their large Stokes shifts and extremely long emission lifetimes (from microseconds to milliseconds) compared to more traditional fluorophores (e.g. fluorescein, allophycoyanin, phycoerythrin, and rhodamine). The biological fluids or serum commonly used in these research applications contain many compounds and proteins which are naturally fluorescent. Therefore, the use of conventional, steady-state fluorescence measurement presents serious limitations in assay sensitivity. Long-lived fluorophores, such as lanthanides, combined with time-resolved detection (a delay between excitation and emission detection) minimizes prompt fluorescence interference. This method (commonly referred to as time-resolved fluorometry or TRF) involves two fluorophores: a donor and an acceptor. Excitation of the donor fluorophore (in this case, the lanthanide ion complex) by an energy source (e.g. flash lamp or laser) produces an energy transfer to the acceptor fluorophore if they are within a given proximity to each other (known as the Förster's radius). The acceptor fluorophore in turn emits light at its characteristic wavelength. The two most commonly used lanthanides in life science assays are shown below along with their corresponding acceptor dye as well as their excitation and emission wavelengths and resultant Stokes shift (separation of excitation and emission wavelengths).

Common lanthanide donor-acceptor pairings

Donor [7] Excitation⇒Emission λ (nm)AcceptorExcitation⇒Emission λ (nm)Stokes Shift (nm)

(donor excitation ⇒ acceptor emission)

Europium 3+340⇒615 Allophycocyanin 615⇒660320
Terbium 3+340⇒545 Phycoerythrin 545⇒575235

TR-FRET example

Spectral overlay of excitation and emission profiles of Europium and Allophycocyanin labeled with Stokes shift and excitation and emission wavelengths to illustrate the separation of wavelengths possible in some TR-FRET assays. EuAPC exem overlay annotated.png
Spectral overlay of excitation and emission profiles of Europium and Allophycocyanin labeled with Stokes shift and excitation and emission wavelengths to illustrate the separation of wavelengths possible in some TR-FRET assays.

As noted in the table above, fluorescent energy transfer from Europium to allophycocyanin can be used in a time resolved manner, particularly in biomolecular screening assays. The figure at right shows the intersection of the emission from Europium with the excitation of allophycocyanin (APC) where energy transfer occurs when Europium and APC are brought into proximity via biomolecular interactions.

When these two fluorophores are brought together by a biomolecular interaction, a portion of the energy captured by the Europium during excitation is released through fluorescence emission at 620 nm, while the remaining energy is transferred to the APC. This energy is then released by APC as specific fluorescence at 665 nm only via FRET with Europium.

Through the design of the high-throughput screening assay, the materials are mixed, and if the enzyme does act on the peptide, all components will bind their respective targets and FRET will occur. [8]

The instrument used to measure the assay then delays the reading of the emitted light by several hundred milliseconds after the incident/excitation light (the light energy pulse supplied by the instrument to excite the donor molecule) in order to eliminate any 'cross-talk' between the excitation and emission signals. ('cross-talk' in this instance refers to overlapping spectral profiles, which could result in false-positives, false-negatives, or reduced sensitivity depending on the assay design. [9] ) This process comprises the 'time-resolved' aspect of the assay.

Related Research Articles

Fluorescence Emission of light by a substance that has absorbed light

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. The most striking example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, while the emitted light is in the visible region, which gives the fluorescent substance a distinct color that can be seen only when exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.

Fluorescence spectroscopy

Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.

Fluorophore Agents that emit light after excitation by light

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 µL per well. Higher density microplates are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.

Förster resonance energy transfer

Förster or fluorescence resonance energy transfer (FRET), resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.

Fluorescence microscope

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

Two-photon excitation microscopy

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of NIR light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching.

IAEDANS Chemical compound

IAEDANS is an organic fluorophore. It stands for 5-({2-[ amino]ethyl}amino)naphthalene-1-sulfonic acid. It is widely used as a marker in fluorescence spectroscopy.

Bimolecular fluorescence complementation

Bimolecular fluorescence complementation is a technology typically used to validate protein interactions. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. Therefore, through the visualisation and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest.


Allophycocyanin is a protein from the light-harvesting phycobiliprotein family, along with phycocyanin, phycoerythrin and phycoerythrocyanin. It is an accessory pigment to chlorophyll. All phycobiliproteins are water-soluble and therefore cannot exist within the membrane like carotenoids, but aggregate, forming clusters that adhere to the membrane called phycobilisomes. Allophycocyanin absorbs and emits red light, and is readily found in Cyanobacteria, and red algae. Phycobilin pigments have fluorescent properties that are used in immunoassay kits. In flow cytometry, it is often abbreviated APC. To be effectively used in applications such as FACS, High-Throughput Screening (HTS) and microscopy, APC needs to be chemically cross-linked.

Quenching (fluorescence)

Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on pressure and temperature. Molecular oxygen, iodide ions and acrylamide are common chemical quenchers. The chloride ion is a well known quencher for quinine fluorescence. Quenching poses a problem for non-instant spectroscopic methods, such as laser-induced fluorescence.

Cyanines, also referred to as tetramethylindo(di)-carbocyanines are defined as "synthetic dyes with the general formula R2N[CH=CH]nCH=N+R2↔R2N+=CH[CH=CH]nNR2 in which the nitrogen and part of the conjugated chain usually form part of a heterocyclic system, such as imidazole, pyridine, pyrrole, quinoline and thiazole." Cyanines are used in industry biotechnology.

Fluorescence anisotropy or fluorescence polarization is the phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarization. Early pioneers in the field include Aleksander Jablonski, Gregorio Weber, and Andreas Albrecht. The principles of fluorescence polarization and some applications of the method are presented in Lakowicz's book.

A dark quencher is a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat; while a typical (fluorescent) quencher re-emits much of this energy as light. Dark quenchers are used in molecular biology in conjunction with fluorophores. When the two are close together, such as in a molecule or protein, the fluorophore's emission is suppressed. This effect can be used to study molecular geometry and motion.

Fluorescence in the life sciences

Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules by means of fluorescence. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.

The FluoProbes series of fluorescent dyes were developed by Interchim to improve performances of standard fluorophores. They are designed for labeling biomolecules, cells, tissues or beads in advanced fluorescent detection techniques.

Single molecule fluorescence resonance energy transfer is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected on a single molecule level. In contrast to "ensemble FRET" which provides the FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium. Heterogeneity among different molecules can also be observed.

Fluorescent glucose biosensors are devices that measure the concentration of glucose in diabetic patients by means of sensitive protein that relays the concentration by means of fluorescence, an alternative to amperometric sension of glucose. Due to the prevalence of diabetes, it is the prime drive in the construction of fluorescent biosensors. A recent development has been approved by the FDA allowing a new continuous glucose monitoring system called EverSense, which is a 90 day glucose monitor using fluorescent biosensors.

Lanthanide probes are a non-invasive analytical tool commonly used for biological and chemical applications. Lanthanides are metal ions which have their 4f energy level filled and generally refer to elements cerium to lutetium in the periodic table. The fluorescence of lanthanide salts is weak because the energy absorption of the metallic ion is low; hence chelated complexes of lanthanides are most commonly used. The term chelate derives from the Greek word for “claw,” and is applied to name ligands, which attach to a metal ion with two or more donor atoms through dative bonds. The fluorescence is most intense when the metal ion has the oxidation state of 3+. Not all lanthanide metals can be used and the most common are: Sm(III), Eu(III), Tb(III), and Dy(III).

Fluorescence imaging

Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.


  1. 1 2 Yan, Y (2003). "Analysis of protein interactions using fluorescence technologies". Current Opinion in Chemical Biology. 7 (5): 635–640. doi:10.1016/j.cbpa.2003.08.017. ISSN   1367-5931.
  2. Periasamy, Ammasi (2005). Molecular Imaging: FRET Microscopy and Spectroscopy (Methods in Physiology Series). Academic Press. p. 336. ISBN   978-0195177206.
  3. Piston, David W.; Kremers, Gert-Jan (2007). "Fluorescent protein FRET: the good, the bad and the ugly". Trends in Biochemical Sciences. 32 (9): 407–414. doi:10.1016/j.tibs.2007.08.003. ISSN   0968-0004. PMID   17764955.
  4. Fu, Haian (2004). Protein-Protein Interactions: Methods and Applications. Humana Press, Inc. p. 544. ISBN   978-1588291202.
  5. Glickman, J. Fraser; Xiang Wu; Robert Mercuri; Chantal Illy; Benjamin R. Bowen; Yang He; Matthew Sills (2002). "A Comparison of ALPHAScreen, TR-FRET, and TRF as Assay Methods for FXR Nuclear Receptors". J Biomol Screen. 7 (1): 3–10. doi: 10.1177/108705710200700102 . PMID   11897050.
  6. Sadler, T. M.; Achilleos, M.; Ragunathan, S.; Pitkin, A.; LaRocque, J.; Morin, J.; et al. (2004). "Development and comparison of two nonradioactive kinase assays for I kappa B kinase". Anal Biochem. 326 (1): 106–13. doi:10.1016/j.ab.2003.11.021. PMID   14769342.
  7. Gschneidner Jr., Karl A. (2007). Handbook on the Physics and Chemistry of Rare Earths, Volume 37: Optical Spectroscopy. North Holland. p. 558. ISBN   978-0444521446.
  8. Degorce, Francois (2009). "HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications". Current Chemical Genomics. 3 (1): 22–32. doi:10.2174/1875397300903010022. ISSN   1875-3973. PMC   2802762 . PMID   20161833.
  9. Thews, Elmar; Margarita Gerken; Reiner Eckert; Johannes Zäpfe; Carsten Tietz; Jörg Wrachtrup (2005). "Cross Talk Free Fluorescence Cross Correlation Spectroscopy in Live Cells". Biophysical Journal. 89 (3): 2069–2076. Bibcode:2005BpJ....89.2069T. doi:10.1529/biophysj.104.057919. PMC   1366709 . PMID   15951373.