Donor Fluorescence Research Articles

Theoretical model of donor–donor and donor–acceptor energy transfer on a nanosphere

In this study, we introduce a novel advancement in the field of theoretical exploration. Specifically, we investigate the transfer and trapping of electronic excitations within a two-component disordered system confined to a finite volume. The implications of our research extend to energy transfer phenomena on spherical nanoparticles, characterized by randomly distributed donors and acceptors on their surface. Utilizing the three-body Padé approximant technique, previously employed in single-component systems, we apply it to address the challenge of trapping within our system. To validate the robustness of our model, we conduct Monte Carlo simulations on a donor–acceptor system positioned on a spherical nanoparticle. In particular, very good agreement between the model and Monte Carlo simulations has been found for donor fluorescence intensity decay.

RSS and ROS Sequentially Activated Carbon Monoxide Release for Boosting NIR Imaging-Guided On-Demand Photodynamic Therapy.

Carbon monoxide shows great therapeutic potential in anti-cancer. In particular, the construction of multifunctional CO delivery systems can promote the precise delivery of CO and achieve ideal therapeutic effects, but there are still great challenges in design. In this work, a RSS and ROS sequentially activated CO delivery system is developed for boosting NIR imaging-guided on-demand photodynamic therapy. This designed system is composed of a CO releaser (BOD-CO) and a photosensitizer (BOD-I). BOD-CO can be specifically activated by hydrogen sulfide with simultaneous release of CO donor and NIR fluorescence that can identify H2S-rich tumors and guide light therapy, also depleting H2S in the process. Moreover, BOD-I generates 1O2 under long-wavelength light irradiation, enabling both PDT and precise local release of CO via a photooxidation mechanism. Such sequential activation of CO release by RSS and ROS ensured the safety and controllability of CO delivery, and effectively avoided leakage during delivery. Importantly, cytotoxicity and in vivo studies reveal that the release of CO combined with the depletion of endogenous H2S amplified PDT, achieving ideal anticancer results. It is believed that such theranostic nanoplatform can provide a novel strategy for the precise CO delivery and combined therapy involved in gas therapy and PDT.

Ultrafast photoactivated nitric oxide release with a pyrene fluorescence probe

Nitric oxide (NO) plays important roles in both physiology and therapeutics. Nonetheless, the in-situ monitoring of NO release from most existing donors is challenging, inhibiting advanced research in both chemistry and biology. In this study, we introduce an innovative photochemotherapeutic agent that incorporates a nitroaniline NO moiety with an integrated pyrene fluorophore. This design enables controlled NO release upon light irradiation, allowing for precise quantification through a dramatic fluorescence enhancement (with a 3000-fold increase in intensity). Ultrafast spectroscopy data revealed that the NO release process is exceptionally rapid, completing within 13.3 ps. Additionally, the donor's fluorescence was successfully utilized for in situ cell imaging upon direct photoexcitation. Consequently, using this fluorescent probe enables the accurate visualization of NO release kinetics, dosage, and localization in real-time. The unique advantages of this NO donor are anticipated to significantly advance research in NO chemistry and biology.

Förster resonance energy transfer efficiency measurements on vinculin tension sensors at focal adhesions using a simple and cost-effective setup.

Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster resonance energy transfer (FRET)-based tension sensors are a remarkable tool for studying these forces and should be made easier to use. We prove that absolute FRET efficiency can be measured on a simple setup, an order of magnitude more cost-effective than a standard FRET microscopy setup, by applying it to vinculin tension sensors (VinTS) at the focal adhesions of live CHO-K1 cells. Our setup located at Université Paris-Saclay acquires donor and acceptor fluorescence in parallel on two low-cost CMOS cameras and uses two LEDs for rapid switching of the excitation wavelength at a reduced cost. The calibration required to extract FRET efficiency was achieved using a single construct (TSMod). FRET efficiencies were measured for VinTS and the tail-less control VinTL, lacking the actin-binding domain of vinculin. Measurements were confirmed on the same cell type using a more standard intensity-based setup located at Rutgers University. The average FRET efficiency of VinTS () over more than 10,000 focal adhesions is significantly lower () than that of VinTL (), our control that is insensitive to force, in agreement with the force exerted on vinculin at focal adhesions. Attachment of the CHO-K1 cells on fibronectin decreases FRET efficiency, thus increasing the force, compared with poly-lysine. FRET efficiency for the VinTL control is consistent with all measurements currently available in the literature, confirming the validity of our measurements and hence of our simpler setup. Force measurements, resolved spatially inside a cell, can be achieved using FRET-based tension sensors with a cost effective intensity-based setup. This will facilitate combining FRET with techniques for applying controlled forces such as optical tweezers.

Physical mechanisms on FRET and ICT for efficient Y6:PM6 as bulk heterojunction active layers.

Organic solar cells (OSCs) have emerged as a promising new energy technology because of their advantages of efficient light harvesting, comprehensive material sources, and flexible and translucent devices. In this study, fluorescence resonance energy transfer (FRET) and intermolecular charge transfer (ICT) in the donor-acceptor system for efficient OSCs of the Y6:PM6 heterostructure are investigated with ultrafast pump-probe transient absorption spectroscopy, time-resolved fluorescence spectroscopy, steady absorption, and fluorescence spectroscopy, which is significantly supported by theoretical results. The physical mechanisms of FRET and ICT in the donor-acceptor system for efficient OSCs of the Y6:PM6 heterostructure are investigated theoretically and experimentally. FRET can decrease the recombination of electron-hole in the donor's fluorescence and enhance the acceptor's fluorescence. Our study contributes to a better understanding of FRET and ICT and provides valuable references for the rational design of FRET- and ICT-based OSCs.

FRET-based carbazole-fluorescein ionic nanoparticle for use as an effective bioimaging agent.

Fӧrster resonance energy transfer (FRET)-based systems are widely applicable in many areas of interest. In this study, a novel FRET-based ionic material (IM) was synthesized by pairing carbazole imidazolium cation (CI+) with fluorescein anion (Fl2-) through a simple ion-exchange method. The resulting IM ([CI]2[Fl]) was converted into an ionic nanoparticle (INP) in aqueous media for practical use for bioimaging application. The photophysical properties of the parent dyes, [CI]2[Fl], and INP were studied in detail. All FRET parameters were calculated in the synthesized material. [CI]2[Fl] exhibited a significant spectral overlap integral and an ideal theoretical FRET distance. The presence of the FRET mechanism was verified by the observed decrease in donor fluorescence lifetime and a moderate FRET efficiency in [CI]2[Fl]. The INP formed from [CI]2[Fl] was evaluated for use as a fluorescent pH probe and bioimaging agent. FRET efficiency of INP is calculated in a series of pH studies which indicates the highest efficiency at physiological pH. Whereas no FRET phenomenon is observed in highly acidic and basic conditions. The pH-dependent photophysical properties of [CI]2[Fl] are monitored and allow for the potential application as a fluorescent probe for the detection of acidic tissues in biological systems. The FRET-capable INP showed superior bioimaging capability in vitro as compared to the parent dye.

Designs and Applications of Multi-stimuli Responsive FRET Processes in AIEgen-Functionalized and Bi-fluorophoric Supramolecular Materials

Materials capable of displaying strong ratiometric fluorescence with Förster resonance energy transfer (FRET) processes have attracted much research interest because of various chemosensor and biomedical applications. This review highlights several popular strategies in designing FRET-OFF/ON mechanisms of ratiometric fluorescence systems. In particular, the developments of organic and polymeric FRET materials featuring aggregation-induced emission-based luminogens (AIEgens), supramolecular assemblies, photochromic molecular switches and surfactant-induced AIE/FRET mechanisms are presented. AIEgens have been frequently employed as FRET donor and/or acceptor fluorophores to obtain enhanced ratiometric fluorescences in solution and solid states. Since AIE effects and FRET processes rely on controllable distances between fluorophores, many interesting fluorescent properties can be designed by regulating aggregation states in polymers and supramolecular systems. Photo-switchable fluorophores, such as spiropyran and diarylethene, provide drastic changes in fluorescence spectra upon photo-induced isomerizations, leading to photo-switching mechanisms to activate/deactivate FRET processes. Supramolecular assemblies offer versatile platforms to regulate responsive FRET processes effectively. In rotaxane structures, the donor-acceptor distance and FRET efficiency can be tuned by acid/base-controlled shuttling of the macrocycle component. The tunable supramolecular interactions are strongly influenced by external factors (such as pH values, temperatures, analytes, surfactants, UV-visible lights, etc.), which induce the assembly and disassembly of host-guest systems and thus their FRET-ON/FRET-OFF behavior. In addition, the changes in donor or acceptor fluorescence profiles upon detections of analytes can also sufficiently alter the FRET behavior and result in different ratiometric fluorescence outputs. The strategies and examples provided in this review offer the insights and toolkits for future FRET-based material developments.

Rapid on-site PEDV detection using homogeneous fluorescence resonance energy transfer-based ELISA

The porcine epidemic diarrhea virus (PEDV) is a major pathogen of swine enteric diseases. Various etiology and serological methods have been employed for PEDV detection, but most of their applications are limited to laboratories. To extend PEDV detection to on-site applications, we design a homogeneous fluorescence resonance energy transfer (FRET)-based enzyme-linked immunosorbent assay (ELISA). Both donor and acceptor fluorescence microspheres modified PEDV antibodies can be linked only to the occurrence of PEDV antigen, thus generating FRET signals, which can be collected by our designed portable FRET immunoassay station (FRETIS). Verified by standard samples, FRET immunoassay reached high sensitivity with a detection limit as TCID50 (median tissue culture infective dose) of 10/mL, which is 10 times more sensitive than colloidal gold test strips; and verified by clinical samples, it was also proved with high accuracy, good selectivity, and repeatability. More importantly, FRET immunoassay could detect PEDV in a 96-well plate in 35 min with only one step of incubation without any further washing steps using field-portable devices and field-operable procedures, well supporting on-site applications. Considering these advantages, this reported FRET immunoassay provides a promising way for multi-sample on-site PEDV detection and can be potentially used in the swine industry.

Cascade Förster Resonance Energy Transfer Studies for Enhancement of Light Harvesting on Dye-Sensitized Solar Cells.

This work reports cascade Förster resonance energy transfer (FRET)-based n-type (ZnO) and p-type (NiO) dye-sensitized solar cells (DSSCs), discussing approaches to enhance their overall performance. Although DSSCs suffer from poorer performance than other solar cells, the use of composites with carbon dot (Cdot) can enhance the power conversion efficiency (PCE) of DSSCs. However, further improvements are demanded through molecular design to stimulate DSSCs. Here, a photosensitized system based on a cascade FRET was induced alongside the conventional photosensitizer dye (N719). To N719 in a DSSC is transferred the energy cascaded through donor fluorescence materials (pyrene, 3-acetyl-7-N,N-diethyl-coumarin or coumarin and acridine orange), and this process enhances the light-harvesting properties of the sensitizers in the DSSC across a broad region of the solar spectrum. PCE values of 10.7 and 11.3% were achieved for ZnO/Cdot and NiO/Cdot DSSCs, respectively. These high PCE values result from the energy transfer among multi-photosensitizers (cascade FRET fluorophores, N719, and Cdot). Moreover, Cdot can play a role in intensifying the adsorption of dyes and discouraging charge recombination on the semiconductor. The present results raise expectations that a significant improvement in photovoltaic performance can be attained of DSSCs exploiting the cascade FRET photonics phenomenon.

Plasmon effect on simultaneous singlet-singlet and triplet-singlet energy transfer

The influence of plasmonic effect of Ag nanoparticles (NPs) on singlet-singlet (S–S) and triplet-singlet (T–S) energy transfer in the same donor–acceptor pair of organic molecules was studied. It was established that a 7-fold increase in the fluorescence and phosphorescence intensity of the energy donor (Rose bengal) and a 4-fold increase in the fluorescence intensity of the acceptor (polymethine dye) are observed on silver island films. The changes are associated with an increase in the emission rate of dye molecules, as evidenced by a decrease in the lifetime of their luminescence in the presence of Ag NPs. An increase in the efficiency of both types of energy transfer was recorded in the plasmon field of Ag NPs. The plasmon effect has almost the same influence on both S–S and T–S energy transfer. Estimation of the energy transfer rates using of the model of electric dipole interaction showed a good correlation between the experimental and calculated data for the S–S energy transfer.

Construction of mitochondria targeted and FRET based ratiometric sensing nanoplatform for sulfur dioxide accurate detection in vitro and in vivo

Sulfur dioxide (SO2) is a key molecule in organisms that is involved in the regulation of different physiological procedures. Aberrant SO2 causes a variety of diseases, such as cancer and neurodegeneration. Thus, sensitive and selective detection of SO2 is of great importance. Based on the Förster resonance energy transfer (FRET) between green fluorescence carbon dots (GCDs) donor and amide-linked near-infrared fluorescence emissive organic small molecular dye (CDDBT) acceptor, one ratiometric fluorescent nano platform, Mito-GCDs-CDDBT for mitochondria SO2 sensing was constructed. In this FRET sensing system, CDDBT served as the receptor for SO2, and the presence of SO2 enhanced GCDs green fluorescence signal and quenched CDDBT near-infrared fluorescence signal due to the disruption of FRET. Mito-GCDs-CDDBT could sensitively detect SO2 with a detection limit of as low as 0.701 μM. Meanwhile, Mito-GCDs-CDDBT achieved fluorescence imaging to measure the response of cellular exogenous and endogenous SO2 with remarkable mitochondrial targeting. Moreover, Mito-GCDs-CDDBT also realized SO2 sensing in vivo including zebrafish and mice. The as-prepared versatile nanoplatform displayed several advantages, such as mitochondria targeting, FRET-based sensitive detection, and sensing capabilities in biological milieu. Potentially, it could be applied in the diagnostics of SO2 involved diseases.

Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro.

In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Förster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target—green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker—were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors.

Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines.

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τD and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E–τD diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines.

New Core-Shell Nanostructures for FRET Studies: Synthesis, Characterization, and Quantitative Analysis.

This work describes the synthesis and characterization of new core-shell material designed for Förster resonance energy transfer (FRET) studies. Synthesis, structural and optical properties of core-shell nanostructures with a large number of two kinds of fluorophores bound to the shell are presented. As fluorophores, strongly fluorescent rhodamine 101 and rhodamine 110 chloride were selected. The dyes exhibit significant spectral overlap between acceptor absorption and donor emission spectra, which enables effective FRET. Core-shell nanoparticles strongly differing in the ratio of donors to acceptor numbers were prepared. This leads to two different interesting cases: typical single-step FRET or multistep energy migration preceding FRET. The single-step FRET model that was designed and presented by some of us recently for core-shell nanoparticles is herein experimentally verified. Very good agreement between the analytical expression for donor fluorescence intensity decay and experimental data was obtained, which confirmed the correctness of the model. Multistep energy migration between donors preceding the final transfer to the acceptor can also be successfully described. In this case, however, experimental data are compared with the results of Monte Carlo simulations, as there is no respective analytical expression. Excellent agreement in this more general case evidences the usefulness of this numerical method in the design and prediction of the properties of the synthesized core-shell nanoparticles labelled with multiple and chemically different fluorophores.

The isoquinoline PRL-295 increases the thermostability of Keap1 and disrupts its interaction with Nrf2

SummaryTranscription factor Nrf2 and its negative regulator Keap1 orchestrate a cytoprotective response against oxidative, metabolic, and inflammatory stress. Keap1 is a drug target, with several small molecules in drug development. Here, we show that the isoquinoline PRL-295 increased Keap1 thermostability in lysates from cells expressing fluorescently tagged Keap1. The thermostability of endogenous Keap1 also increased in intact cells and murine liver following PRL-295 treatment. Fluorescence Lifetime Imaging–Förster Resonance Energy Transfer (FLIM-FRET) experiments in cells co-expressing sfGFP-Nrf2 and Keap1-mCherry further showed that PRL-295 prolonged the donor fluorescence lifetime, indicating disruption of the Keap1-Nrf2 protein complex. Orally administered PRL-295 to mice activated the Nrf2transcriptional target NAD(P)H:quinone oxidoreductase 1 (NQO1) in liver and decreased the levels of plasma alanine aminotransferase and aspartate aminotransferase upon acetaminophen-induced hepatic injury. Thus, PRL-295 engages the Keap1 protein target in cells and in vivo, disrupting its interaction with Nrf2, leading to activation of Nrf2-dependent transcription and hepatocellular protection.

Oligomerization of yeast α-factor receptor detected by fluorescent energy transfer between ligands

G-protein-coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive evidence that GPCRs exist as homo-and hetero-oligomeric complexes; however, in many cases, the role of oligomerization and the extent to which it occurs at low physiological levels of receptor expression in cells remain unclear. We report here the use of flow cytometry to detect receptor-receptor interactions based on fluorescence resonance energy transfer between fluorescently labeled cell-impermeant ligands bound to yeast α-mating pheromone receptors that are members of the GPCR superfamily. A novel, to our knowledge, procedure was used to analyze energy transfer as a function of receptor occupancy by donor and acceptor ligands. Measurements of loss of donor fluorescence due to energy transfer in cells expressing high levels of receptors were used to calibrate measurements of enhanced acceptor emission due to energy transfer in cells expressing low levels of receptors. The procedure allows determination of energy transfer efficiencies over a 50-fold range of expression of full-length receptors at the surface of living cells without the need to create fluorescent or bioluminescent fusion proteins. Energy transfer efficiencies for fluorescently labeled derivatives of the receptor agonist α-factor do not depend on receptor expression level and are unaffected by C-terminal truncation of receptors. Fluorescently labeled derivatives of α-factor that act as receptor antagonists exhibit higher transfer efficiencies than those for labeled agonists. Although the approach cannot determine the number of receptors per oligomer, these results demonstrate that ligand-bound, native α-factor receptors exist as stable oligomers in the cell membranes of intact yeast cells at normal physiological expression levels and that the extent of oligomer formation is not dependent on the concentration of receptors in the membrane.

Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans.

The force-induced unfolding and refolding of proteins is speculated to be a key mechanism in the sensing and transduction of mechanical signals in the living cell. Yet, little evidence has been gathered for its existence in vivo. Prominently, stretch-induced unfolding is postulated to be the activation mechanism of the twitchin/titin family of autoinhibited sarcomeric kinases linked to the mechanical stress response of muscle. To test the occurrence of mechanical kinase activation in living working muscle, we generated transgenic Caenorhabditis elegans expressing twitchin containing FRET moieties flanking the kinase domain and developed a quantitative technique for extracting FRET signals in freely moving C. elegans, using tracking and simultaneous imaging of animals in three channels (donor fluorescence, acceptor fluorescence, and transmitted light). Computer vision algorithms were used to extract fluorescence signals and muscle contraction states in each frame, in order to obtain fluorescence and body curvature measurements with spatial and temporal precision in vivo. The data revealed statistically significant periodic changes in FRET signals during muscle activity, consistent with a periodic change in the conformation of twitchin kinase. We conclude that stretch-unfolding of twitchin kinase occurs in the active muscle, whereby mechanical activity titrates the signaling pathway of this cytoskeletal kinase. We anticipate that the methods we have developed here could be applied to obtaining in vivo evidence for force-induced conformational changes or elastic behavior of other proteins not only in C. elegans but in other animals in which there is optical transparency (e.g., zebrafish).

Kinetics of protein-assisted nucleic acid interconversion monitored by transient time resolved fluorescence in microfluidic droplets.

Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(–) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (–)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.

Förster Energy Transfer in Core–Shell Nanoparticles: Theoretical Model and Monte Carlo Study

The model of Förster excitation energy transfer on a spherical core–shell nanoparticle was presented. A general expression for fluorescence intensity decay was obtained for an arbitrary number of acceptors linked chemically to the shell. It was found that the dynamical behavior of the system is extremely sensitive to the number of acceptors and the size of the nanoparticle. Monte Carlo simulations performed for the energy transfer parameters taken from an independent experiment show excellent agreement with the model for donor fluorescence decay and its mean lifetime. The original model was then extended to the common experimental case of core–shell nanoparticle size distribution, assuming the Gaussian distribution function of their radii. This effect leads to slower fluorescence decays and longer mean fluorescence lifetimes, as revealed by Monte Carlo simulations.

The fluorescence quantum yield parameter in Förster resonance energy transfer (FRET)—Meaning, misperception, and molecular design

The fluorescence quantum yield parameter in Förster resonance energy transfer (FRET) processes underpins vital phenomena ranging from light harvesting in photosynthesis to design of sensors for monitoring physiological processes. The criteria for choosing a donor for use in FRET processes include chemical features (solubility, bioconjugatability, synthetic accessibility, and stability) as well as photophysical properties pertaining to absorption (wavelength and molar absorption coefficient) and fluorescence (wavelength and fluorescence quantum yield). The value of the donor fluorescence quantum yield (Φf, or emphatically, Φf(D)) alone has sometimes been thought (erroneously) to place a ceiling on the possible quantum yield of energy transfer (Φtrans). A high value of the donor Φf, while attractive, is not at all essential; indeed, many valuable candidates for use as FRET donors have likely been excluded on the basis of this injudiciously applied filter. Such disregard is unwarranted. In this tutorial overview, the equations for FRET are reviewed along with pertinent core concepts in photophysics. An analogy using simple hydraulics provides a pedagogical tool for the non-aficionado to better understand photochemical kinetics. Ten examples are presented of donor–acceptor systems with donors that exhibit a range of Φf values (0.60, 0.59. 0.21, 0.17, 0.12, 0.118, 0.04, 0.018, 0.007, and 0.003; i.e., 60%–0.3%), yet for each corresponding donor–acceptor pair, the value of Φtrans is at least 0.70 and in some cases nearly 1.00 (i.e., 70%–100%). The systems encompass protein, synthetic inorganic, and synthetic organic architectures. The objectives of this illustrative review are to deepen understanding of FRET and to broaden molecular design considerations by enabling selection from among a far richer set of donors for use in FRET processes.