W. Russ Algar

Förster resonance energy transfer (FRET) is a versatile part of the toolbox of fluorescence methods. This through-space, photon-less energy transfer process between a donor fluorophore and an acceptor chromophore is perhaps most famous for its utility as a “molecular ruler” that can resolve nanometer-scale distances. FRET is also a popular and advantageous basis for biomolecular assays and sensors.

One of the most attractive features of the Förster resonance energy transfer (FRET) mechanism is that it can be measured with the simplest of fluorimeters up through to the most advanced single-molecule fluorescence systems. However, this relative simplicity sometimes belies careful considerations that must be taken into account in most FRET experiments. As the user base for FRET and the scope of its applications continues to grow, it is important to understand how to design an optimized and rigorous FRET experiment. This article provides a brief overview of the key considerations, most of which are linked to Figure 1. A much more detailed and broader scope of guidance can be found in the literature (1), and readers are encouraged to review the fundamental FRET theory in the literature (2,3).

FIGURE 1: Conceptual illustration of FRET. Parts of this illustration are linked to the discussion in the text through lowercase letter labels (a–j).

Although basic FRET theory can be found in numerous publications and on a multitude of websites, these basics are not always sufficient. For example, the classic equation (shown in Figure 1a) for FRET efficiency (the probability of FRET occurring) applies to a uniform ensemble of discrete one donor–one acceptor pairs. This equation must be modified if multiple acceptors interact with a common donor, and will often be a poor approximation if multiple donors interact with a common acceptor, if a system is highly heterogeneous in its donor–acceptor combinations, or if different FRET pathways compete with one another. Such non-classic systems will need to be modeled using analyses that compare the rates of the various FRET pathways against one another and the rates of other excitation and relaxation processes (4).

Furthermore, many sources present the equation for the FRET-relevant spectral overlap integral (between donor emission and acceptor absorption) without specifying units for the quantities, which must be correct for downstream estimation of the Förster distance, 

 (Figure 1b). Units should thus be confirmed in a reference source or assessed by dimensional analysis. Most estimations of a Förster distance will also assume a “dynamic averaging regime” with an orientation factor of 

 = 2/3 between donor and acceptor (Figures 1b, 1c). A recent perspective has addressed potential challenges from this assumption in detail (5). Adopting 

 = 2/3 or another “special” value (such as, for example, static averaging regime) is not necessarily a bad practice, provided the uncertainties associated with this assumption are factored into drawing conclusions from an experiment—a conclusion predicated on an accurate and precise value for 

Define the Mechanism of Donor-Acceptor Proximity

An essential requirement of FRET is close proximity between donor and acceptor, usually within 10 nm, but up to approximately 15 nm in special cases. A rigorous FRET experiment will rationally design or, at minimum, understand the origin of donor-acceptor proximity (Figure 1d). Common approaches include having the donor and the acceptor attached to the same macromolecular scaffold, and also having molecules that are separately labeled with donor and acceptor interacting through specific binding, aggregation, or assembly on a common scaffold or surface.

Match the Measurement Modality and FRET Metric to the Objective and Limitations of the Experiment 

FRET is famously useful as a “molecular ruler” because the energy transfer efficiency can be used to measure the nanometric distance between donor and acceptor. For such measurements, it is important that the FRET efficiency has maximum variation around the distance of interest. This means avoiding too close an approach to the plateaus in FRET efficiency near 0 and 100%. A mathematical model of FRET efficiency versus distance for a hypothetical system will indicate a range over which efficiency is approximately linear with distance, although the overall relation is sigmoidal.

FRET is also a useful mechanism for sensors. For this application, it is often preferred that the FRET efficiency switches between a negligible value (~0%) and a significant value upon detection of the target analyte (or otherwise maximizes the relative change in the measured FRET signal). This signal change also has a role in determining the concentration of FRET probe that should be used. For example, if it is possible to detect the switch in FRET efficiency for one probe out of 100, then the concentration of probe should not exceed 100-fold of the lowest concentration of target analyte to be detected.

A FRET measurement must also be designed around the available technical capabilities. Important considerations are as follows: whether the metric for assessing FRET will be based on fluorescence intensity or lifetime; whether this metric will be measured for the donor, the acceptor, or both (as applicable); and whether resolution of donor and acceptor emission will be via emission filters or spectrally via monochromator (Figure 1e). For the latter, spectral measurements allow more robust correction of overlapping donor and acceptor fluorescence emission and may thus accommodate a wider array of potential donor–acceptor pairs. Crosstalk between detection channels (wavelengths or filters) for donor and acceptor emission is common and should be checked for, and may require mathematical correction to avoid skewing the value of the chosen FRET metric.

Ensemble FRET efficiency is best reserved as a metric for homogeneous systems. The apparent ensemble efficiency for heterogeneous systems is a function of both the real FRET efficiencies and fractions of whatever subpopulations are in the system. Thus, the ensemble efficiency for a heterogeneous system loses meaning without independent knowledge of the sub-populations. For example, sensing applications detect initially unknown concentrations of the analyte and are better suited to a metric such as donor intensity or lifetime, acceptor intensity, or an acceptor–donor intensity ratio.

Donor and acceptor fluorescence intensities are simple and versatile metrics, but have the drawbacks of being sensitive to the concentration of the FRET pair, measurement volume, and drift or fluctuation in instrumentation. The acceptor–donor intensity ratio and donor fluorescence lifetime are more robust metrics because they are approximately insensitive to sample volume and concentration over whatever range the donor–acceptor proximity is unaffected by concentration. An acceptor–donor intensity ratio and donor fluorescence lifetime are also either self-correcting or less affected by many forms of instrumental drift or fluctuation. Many interesting applications of FRET have a biological system as the gatekeeper of a number of FRET probes being measured (not well-controlled by the experimentalist), making this advantage significant.

Another consideration is whether an ensemble measurement is satisfactory or if single-molecule measurements are necessary. Single-molecule measurements are most informative when heterogeneity or asynchronous processes within a sample are of interest, but are beyond the scope of this short article.

The first and best-known criterion for a good donor–acceptor pair is substantial overlap between the donor emission spectrum and the acceptor absorption spectrum (Figure 1f). However, the considerations do not stop here. The magnitude of the Förster distance must be commensurate with the experimental objective and the mechanism of donor–acceptor proximity. Moreover, the labeling chemistries, solubility, charges, and sizes of the donor and acceptor should also be good matches to the mechanism of proximity. For a FRET pair where both donor and acceptor are fluorescent, it is also preferable that there is an excitation wavelength for the donor at which direct excitation of the acceptor is negligible (Figure 1g). The choices of donor and acceptor must also align with available technical capabilities (such as satisfactory excitation wavelengths and emission filters, suitable resistance to photobleaching). For all of the foregoing, there are pros and cons for the many candidate donor and acceptor materials available, which range from organic dyes to fluorescent proteins to various nanoparticles. One should go into a FRET experiment anticipating that multiple FRET pairs may need to be tried to achieve optimum results.

Some general challenges with fluorescence include the potential sensitivity of the fluorescence intensity of a dye to its local microenvironment (such as viscosity or other restriction of movement, solute concentrations, and more), the lack of a standardized unit for fluorescence intensity, and potentially large differences in brightness between different fluorophores. Consequently, control experiments that ensure that changes in donor and acceptor emission arise from FRET and not from other processes are essential. With only a few exceptions, experimental samples and control samples must usually be measured under the same instrument and with the same settings.

The minimum control sample is a no-FRET reference state (Figure 1h) with only the donor, but more control samples would be beneficial. For example, reabsorption of the donor emission by an acceptor could give the appearance of donor quenching and acceptor sensitization, but is not actually FRET. A control experiment that has a combination of donor and acceptor but with disrupted proximity (Figure 1d) will guard against such trivial energy transfer via reabsorption. If the mechanism of proximity is sufficiently robust, consistent FRET metrics over a range of dilutions may also be a guard against trivial energy transfer.

For a fluorescent acceptor, another important control is a sample with only an acceptor to distinguish between FRET-sensitized acceptor emission and directly excited acceptor emission (Figure 1i). Potential brightness differences mean that the latter cannot be reliably assumed to be negligible simply from qualitative inspection of an absorption spectrum.

It is also good practice to ensure that two metrics yield similar FRET efficiencies for samples expected to have maximum FRET. Commonly, the decreases in donor intensity and lifetime (relative to a no-FRET reference state) are compared (Figure 1j). Commensurate values will tend to indicate an absence of trivial energy transfer. Even when not the preferred metric for all the experiments, a one-time measurement of lifetime may help to confirm FRET. Alternatively (or in addition), quantitative comparison of a measured FRET metric to a predicted one can be diagnostic, as a large discrepancy may indicate something other than FRET (or a poor model of an actual FRET system).

Ensemble FRET measurements are both powerful and accessible to a broad array of experimentalists. Although it surprisingly easy to be fooled into a false observation of FRET, it does not take much additional effort or resources to safeguard against false observations. Again, this article is intended to be a starting point—there are certainly details and special cases beyond the scope of this text. Nevertheless, the above guidance is a solid foundation for many applications of FRET.

(1) W.R. Algar, N. Hildebrandt, S.S. Vogel, and I.L. Medintz, Nat. Meth. 16, 815–829 (2019). 

(2) N. Hildebrandt, C.M. Spillmann, W.R. Algar, T. Pons, M.H. Stewart, E. Oh, K. Susumu, et al, 

FRET – Förster Resonance Energy Transfer

, I.L. Medintz and N. Hildebrandt, Eds. (Wiley-VCH Verlag GmbH & Co., Weinheim, Germany, 2013), pp. 23–62.

(4) M. Massey, H. Kim, E.M. Conroy, and W.R. Algar, 

 is with the Department of Chemistry at the University of British Columbia, in Vancouver, British Columbia, Canada. Direct correspondence to: 

Articles

Key Steps to Follow in a FRET Experiment

We’d like to take a moment to celebrate the 36-year career of Ulrich J. Krull, on the occasion of his retirement earlier this year. Ulli is an outstanding example of a researcher, educator, and leader in the field of analytical chemistry who has also been an enthusiastic and tireless driver of academic partnerships with industry, government, and communities.

Ulrich J. Krull in 2017. Photo credit: Lisa Sakulensky.

Ulli was born in 1956 in Berlin, Germany, and immigrated to Canada with his family in 1959. Raised in the greater Toronto area, Ulli met Carol—his high-school sweetheart, wife of 41 years and counting, and greatest supporter—at age 16. Ulli obtained his BSc, MSc, and PhD degrees from the University of Toronto between 1975 and 1983. In 1985, he joined the University of Toronto’s Mississauga campus (UTM) as an Assistant Professor. At this time, UTM was a campus of 3500 full-time and part-time students and had two main buildings for teaching and research.

The initial work in Ulli’s research group focussed on the development of artificial membranes for chemical sensing applications—specifically, how interfacial perturbations (such as changes in pH) from selective binding interactions could alter the structure of an underlying artificial lipid bilayer membrane. These alterations in membrane structure were transduced by environmentally sensitive fluorophores embedded within the membranes and detected spectroscopically (1,2). A subsequent transition from liquid-supported membranes to solid-supported membranes was Ulli’s first foray into fiber-optic chemical sensor development, which would continue for many years (3).

The first of many leadership roles at UTM came in 1994, when Ulli became the Associate Dean of Sciences. His first challenge—at the request of the Mayor of Mississauga, Hazel McCallion—was to devise a mechanism to build a “healthy city” by leveraging the expertise and advances at UTM for the benefit of the civic community. With the engagement of UTM students, faculty, and staff, Ulli formulated a plan that resulted in the formation of the UTM-City of Mississauga Healthy City Stewardship Centre. This centre fostered a city of people with physical, mental, and spiritual health through the provision of equal access to information and services, maintenance of a clean and sustainable environment, and the establishment of a supportive community culture and public services. This effort was ultimately recognized with the World Leadership Award in 2006, and the mayor enjoyed another 20 consecutive years in office before retiring. By this point, UTM had grown to approximately5000 full-time and part-time students and saw its first major infrastructure expansion with the opening of a third academic building in 1992.

Inspired by the initiation of the human genome project, the early 1990s saw Ulli’s research program pivot to the development of fluorimetric fiber optic DNA biosensors for the detection pathogenic bacteria and single nucleotide polymorphisms associated with human disease. His group quickly established their place among the leaders in this field, with seminal papers amassing over 500 citations to date, as per citation counts (08/2021) for individual papers from Google Scholar, and career citation counts from Web of Science (4,5). Further research saw innovations in interfacial chemistry, including refractive index matching between immobilized oligonucleotide films and underlying waveguides to bypass evanescent fields and obtain direct excitation and high-efficiency recovery of fluorescence (6), and fundamental studies and new methods for using high oligonucleotide probe densities to optimize binding selectivity (7). Much of this research was bookended by the McBryde Medal (1994) and Maxxam Award (2002), the two highest honors for analytical chemistry research in Canada. During this same period, Ulli was also named the AstraZeneca Chair in Biotechnology, won a Teaching Excellence Award at UTM, began his 20-year tenure as an editor for 

, and won the University of Toronto’s premier tri-campus honor for excellence in research, teaching, and service.

The new millennium brought many new leadership challenges and opportunities for Ulli, including terms as Vice Principal of Research (2003–13), Vice Dean graduate (2006–08), Vice Principal of Special initiatives (2012–16), and, ultimately, Principal of UTM and Vice President of the University of Toronto (2017–20). Over these years, Ulli led the creation of the Master of Biotechnology Program (2001), the Centre for Applied Biosciences & Biotechnology (2003), the Western Greater Toronto Area Convergence Centre (2005; technology commercialization and university-industry-government networking), the Research Innovation Commercialization Centre (2009; a not-for-profit innovation hub and business incubator service), the Mississauga Academy of Medicine (2011), the Institute for Management and Innovation (2014; a professional school bridging business with health and science), the Continuum Robotics Laboratory (2019), and a new science building that is now under construction. The new science building is the largest capital project initiated to date across all three campuses of the University of Toronto.

Ulli’s vision and dedication has been rewarded with a thriving UTM campus that now boasts over 15,000 undergraduate students, 860 graduate students, 1200 permanent faculty and staff, and 9 academic and research buildings. It is no surprise that Ulli won the Paul W. Fox Award (2011) from the UTM Alumni Association for distinguished voluntary service and promotion of the university and was named one of the 50 most influential people in the 50-year history of UTM (2017). As a further tribute to his accomplishments in research, innovation and in citizenship, the Ulrich J. Krull Research Excellence Award was established in 2020 to recognize students who combine outstanding research with leadership in entrepreneurship or community engagement (8).

Even with his many leadership roles, Ulli maintained a vibrant research program. Research toward the design of surface chemistry to optimize nucleic acid binding selectivity continued with mixed films of polymers and oligonucleotides, strategies were explored for the use of tethered intercalating dyes for DNA biosensor development, and microfluidics (9,10) and luminescent nanomaterials were added as tools for the development of nucleic acid biosensors. The latter included some of the earliest explorations of the use of semiconductor quantum dots and lanthanide-based upconversion nanoparticles for the detection of nucleic acids by energy transfer, as well as fundamental investigation and optimization of nanoparticle interfacial chemistry. Some of this nanoparticle research has been cited over 500 times (11–14).

Over his career, Ulli published more than 270 research articles, accrued nearly 7300 citations, generated more than a dozen patents, was involved with multiple start-up biotechnology ventures, supervised 51 graduate theses, helped organize 45 conferences and symposia, held 17 editorial roles, gave more than 400 invited lectures, attracted or co-attracted $30M CAD in research and infrastructure funding, and was recognized by 28 awards and honors for excellence in research, teaching, and service. His research toward selective biosensor technologies, and his recognition of the importance of interfacial chemistry in this endeavor, will continue to resonate in the field for years to come.

The foregoing summary is a glimpse into the energy and achievements of Ulli Krull, scientist and leader, but we cannot begin to convey the personal impact he had on his communities. His ambition, inventiveness, and dedication has touched the lives of countless people across the city of Mississauga and the whole of Southern Ontario. His humility and kind and enthusiastic teaching and mentorship has inspired innumerable students and colleagues. He is respected from coast-to-coast in the scientific community in Canada and throughout the international analytical chemistry community. He also engaged and supported a myriad of people through more than 25 years as an instructor with the UTM Judo Club, activities with Scouts Canada, community and school outreach events, patronage of The Riverwood Conservancy, and membership in the Royal Astronomical Society of Canada.

Although Ulli’s colleagues and communities will miss him, they are all excited for this next and well-deserved chapter in his life. Four generations of Ulli’s loving family and many good friends are looking forward to spending more time with him.

Ulli, thank you for all that you have done to enrich our lives. We wish you all the best in retirement.

With apologies to all authors whose publications we did not have space to cite.

(1) U.J. Krull, J.D. Brennan, R.S. Brown, S. Hosein, B.D. Hougham, E.T. Vandenberg, 

(2) J.D. Brennan, R.S. Brown, C.P. McClintock, U.J. Krull. 

(3) U.J. Krull, R.S. Brown, B.D. Hougham, I.H. Brock. 

(4) P.A.E. Piunno, U.J. Krull, R.H.E. Hudson, M.J. Damha, H. Cohen, 

(5) P.A.E. Piunno, U.J. Krull, R.H.E. Hudson, M.J. Damha, H. Cohen. 

(6) U.J. Krull, P.A.E. Piunno, R.H.E. Hudson, A. Uddin, M. Damha, US Patent 6,503,711, Jan. 2003.

(7) J.H. Watterson, P.A.E. Piunno, C.C. Wust, U.J. Krull, 

(8) https://www.utm.utoronto.ca/support-utm/student-support/ulrich-j-krull-research-excellence-award

(10) D. Erickson, X. Liu, R. Venditti, D. Li, U.J. Krull, 

A celebration of the 36-year career of Ulrich J. Krull, on the occasion of his retirement earlier this year.

Leading by Example: A Retrospective on the Career of Ulrich J. Krull 

	Fluorescence quenching is the nonradiative loss of excitation energy from a fluorophore through an interaction with another species, called a quencher. It is a powerful, versatile, and widely used methodology that can, for example, be the basis for chemical and biological assays and sensors, and is useful for probing biomolecular structure. A side effect of this broad scope and utility, combined with the relative technical ease of the experiments, is that the analysis, interpretation, and discussion of fluorescence quenching data is sometimes prone to errors. A correct molecular and spectroscopic interpretation is essential for obtaining useful physicochemical insight from quenching data. This article summarizes several common errors to avoid in the consideration of fluorescence quenching data.

Implying That Dynamic Quenching and Static Quenching Are Specific Mechanisms of Quenching

 are not mechanisms of fluorescence quenching, but rather two classes of mechanisms of quenching. Per the recommendations of the International Union of Pure and Applied Chemistry (1), 

is a process that interferes with emission from the excited state of a fluorophore after the excited state has formed. A hallmark of dynamic quenching is thus a change in the observed fluorescence lifetime of the fluorophore. In contrast, 

 is a process that inhibits the initial formation of the emissive excited state, and there is no apparent change in the fluorescence lifetime. Given that neither terminology implies any other details about how quenching occurs, multiple mechanisms of quenching fit the definitions of both terms.

Equating Collisional Quenching and Dynamic Quenching

	Many resources use the quenching of a fluorophore by dissolved oxygen, acrylamide, or halide ions as an illustrative example of dynamic quenching. These instances are also often used as a concurrent example of collisional quenching. The terms 

have thus, unfortunately, become synonymous for some scientists. There are two points that readily debunk this misconception.

	First, there is dynamic quenching that is not collisional quenching. Common examples are Förster resonance energy transfer (FRET) systems, where the fluorophore and quencher are attached to the same molecule (or molecular complex), and thus diffuse together. The energy transfer occurs through space, over distances up to approximately 10 nm, and therefore does not require collisions or any other molecular contact (2). FRET is an additional nonradiative decay pathway from the excited state of a fluorophore and thus shortens its measured fluorescence lifetime.

	Second, multiple mechanisms of quenching have been proposed for well-known collisional quenchers of fluorescence. For example, iodide is thought to quench via promotion of intersystem crossing (ISC) during a collision with a fluorophore (3), oxygen is thought to quench via exciplex formation (among other mechanisms) (4,5), and acrylamide has been proposed to quench via an electron transfer mechanism (6).

	In summary, collision-driven mechanisms of quenching are dynamic quenching, but not all dynamic quenching is collision-mediated quenching.

Equating Static Quenching and Association between Fluorophore and Quencher

	Association between a fluorophore and a quencher does not equate to a static quenching mechanism. FRET, as noted earlier, causes dynamic quenching, and FRET configurations frequently have the fluorophore and quencher associated with one another, even if indirectly through one or more molecules on which they are labels (2). Likewise, systems of fluorophore and quencher pairs that engage in photoinduced electron transfer (PET) are often intimately associated with one another (sometimes as parts of the same molecule) because of the short-range nature of this dynamic quenching mechanism (7). Conceptually, Perrin quenching, which occurs all-or-none inside or outside a sphere of action, is a static quenching mechanism that requires close proximity but not physical association (5,8). That said, Perrin quenching is a phenomenological description, and often arises in a viscous solution or solid matrix from a dynamic quenching mechanism that is very efficient but only at very short range; for example, electron transfer (9). An example of static quenching that is based on association is when a ground state fluorophore and a quencher form a non-fluorescent or weakly-fluorescent complex, as observed with some fluorophores and nucleotides (5,10).

	In sum, not all static quenching requires association between quencher and fluorophore, and not all association between quencher and fluorophore equates to static quenching.

Using FRET as a Generic Term for Quenching

	A surprisingly common misconception (or deduction) is that any fluorescence quenching that is neither collisional quenching nor static quenching must be FRET. The FRET terminology is not generic. Rather, it refers to a specific mechanism of energy transfer to which particular conditions and assumptions are inherent. Although there are many more nuances to FRET theory (2), the basic checklist for considering FRET as a plausible quenching mechanism is a short one. This checklist includes: a defensible expectation of an approximately 1–10 nm separation between fluorophore and quencher; overlap between the fluorophore's emission spectrum and quencher's absorbance spectrum; and a decrease in the fluorescence lifetime of the fluorophore that parallels its quenching. Note that close contact between a fluorophore and a quencher may favor other mechanisms of energy transfer (ET), such as Dexter ET.

Conferring Too Much Diagnostic Value to a Stern-Volmer Plot

 refer to the measured fluorescence intensity for a fixed amount of fluorophore in the absence and presence, respectively, of a certain concentration of quencher, [

], with a linear trend often anticipated.

	A Stern-Volmer plot is an elegant method of determining quantitative information (for example, rate constant) about fluorescence quenching when the nature of that quenching is reasonably well known; however, on its own, a single Stern-Volmer plot is unreliable for identifying how unknown quenching is mediated. The reason is that a Stern-Volmer plot may be linear or curved for multiple reasons (see Figure 1). A collisional quenching process and the formation of a non-fluorescent ground-state complex will both frequently yield straight lines, and curved plots can occur for concurrent quenching by multiple quenching processes, as well as for special cases of single processes; see references (5,8) for discussion.


	Figure 1: Diagram of generic Stern-Volmer plots based on fluorescence intensity. Linear plots, concave-up plots, and concave-down plots (to a plateau) arise from various ways in which quenching is mediated.

	Given the above, the main diagnostic utility of a Stern-Volmer plot is not the qualitative trend of the plot, but rather the value of the quantitative parameter extracted from fitting the data to a quenching model. If this value is not physically reasonable, then the model of quenching likely needs to be reconsidered.

Neglecting to Rigorously Validate How Quenching Is Mediated

 refers to processes that alter the radiative or nonradiative relaxation rates of a fluorophore (for example, intersystem crossing, FRET, electron transfer, proton transfer, H-dimerization). As most quenching mechanisms have a distance dependence, 

 refers to how the necessary proximity is generated between fluorophore and quencher. For example, diffusion mediates collisional quenching, intermolecular forces mediate formation of a non-fluorescent H-dimer, and biomolecular binding interactions frequently mediate FRET (see Figure 2).


	Figure 2: Flowchart that distinguishes between mediation and mechanism of fluorescence quenching, and classifies the mechanisms as dynamic, static, or trivial quenching. The flowchart is representative rather than exhaustive in its content. Note: Sphere-of-action quenching is an unusual case that mimics static quenching (no change in fluorescence lifetime) but may have an underlying dynamic quenching mechanism.

	Trends in quenching efficiencies and, when applicable, the slopes or curvature of Stern-Volmer plots measured under multiple conditions, have diagnostic utility for determining how unknown fluorescence quenching is mediated. For example, changes in temperature or viscosity alter diffusion rates, changes in temperature or solvent polarity alter the value of complexation equilibrium constants, and adding competitive inhibitors, denaturants, or hydrolytic reagents can disrupt binding interactions. The opposite responses of diffusion and complexation equilibria to temperature are often cited to illustrate how these experiments can be diagnostic, but nearly any hypothesized mediation of quenching can be tested through judicious changes in conditions or additives.

	The above experiments are also good guards against trivial quenching, also known as 

. One common inner-filter effect is when the quencher reduces the excitation light available to be absorbed by the fluorophore, and another is when a quencher absorbs light emitted from the fluorophore as fluorescence (11). Progressive shifts in emission spectra that accompany increases in quencher concentration can be diagnostic of inner-filter effects (spectra generally quench uniformly for non-trivial quenching), and fluorescence lifetime measurements are often immune to inner-filter effects. Additives or changes in conditions that disrupt mediation of quenching are also diagnostic because they alter quenching efficiency without altering quencher concentration. If the expected recovery of fluorescence is not observed when the hypothesized mediation of quenching is disrupted, then either the hypothesis needs to be revised, or the quenching is actually trivial.

Undervaluing Absorbance Spectra, Excitation Spectra, and Fluorescence Lifetime Data

	Emission spectra are predominantly used to measure fluorescence quenching. Useful insights can be overlooked by neglecting to also measure absorbance spectra and fluorescence excitation spectra for fluorophore-quencher mixtures. For example, dynamic quenching mechanisms do not affect the light absorption process. Absorbance spectra thus remain unchanged and useful for quantifying the concentration of fluorophores, which is both diagnostic and helpful in avoiding erroneous interpretation of inadvertent changes in fluorophore concentration as quenching. In contrast, the formation of a ground-state non-fluorescent complex between a fluorophore and a quencher may cause changes in absorbance spectra that do not propagate to excitation spectra. Shifts or changes in the shape of excitation spectra are sometimes diagnostic of an excitation-based inner-filter effect. For instances of FRET where the fluorophore of interest (donor) is quenched by another fluorophore (acceptor), the energy transfer leads to emission from the acceptor. An excitation spectrum is useful for assessing what fraction of the measured acceptor emission is from energy transfer rather than from direct excitation (the alternative is measurement of control samples with only the fluorescent acceptor).

	Last, but not least, fluorescence lifetime measurements are almost imperative to deducing an unknown mechanism of quenching. There is no other direct method of establishing dynamic quenching (other parameters that depend on fluorescence lifetime, such as fluorescence anisotropy, can sometimes be substituted as indirect measurements). Discrepancies between lifetime- and intensity-derived quenching efficiencies or Stern-Volmer plots are thus diagnostic of static mechanisms of quenching, or mixed static and dynamic mechanisms, or an inner filter effect alongside quenching.

	In summary, a robust and meaningful analysis, interpretation, and discussion of fluorescence quenching is developed through multiple sets of measurements, attention to both the mechanism and mediation of quenching, precise use of terminology, and knowledge of the hallmarks and nuances of different quenching behaviors.

	(2) N. Hildebrandt, C.M. Spillmann, W.R. Algar, T. Pons, M.H. Stewart, E. Oh, K. Susumu, S.A. Díaz, J.B. Delehanty, and I.L. Medintz, 

	(3) A. Chmyrov, T. Sandén, and J. Widengren, 

	(4) K. Kikuchi, C. Sato, M. Watabe, H. Ikeda, Y. Takahashi, and T. Miyashi, 

 3rd ed. (Springer US, New York, New York, 2006).

	(6) M.R. Eftink, T.J. Selva, and Z. Wasylewski, 

	(7) S. Doose, H. Neuweiler, and M. Sauer, 

Molecular Fluorescence: Principles and Applications

 (Wiley-VCH Verlag GmbH, Weinheim, 2001).

	(10) D.A. Dunn, V.H. Lin, and I.E. Kochevar, 

 are with the Department of Chemistry at the University of British Columbia, in British Columbia, Canada. Direct correspondence to: 

W. Russ Algar

Articles

Key Steps to Follow in a FRET Experiment

Leading by Example: A Retrospective on the Career of Ulrich J. Krull

Key Errors to Avoid in the Consideration of Fluorescence Quenching Data