Förster Resonance Energy Transfer Efficiency Research Articles

Specific FRET Probes Sensitive to Chitosan-Based Polymeric Micelles Formation, Drug-Loading, and Fine Structural Features.

Förster resonance energy transfer (FRET) probes are a promising tool for studying numerous biochemical processes. In this paper, we show the application of the FRET phenomenon to observe the micelle formation from surfactants, micelles self-assembling from chitosan grafted with fatty acid (oleic-OA, or lipoic-LA), cross-linking of SH groups in the micelle's core, and inclusion and release of the model drug cargo from the micelles. Using the carbodiimide approach, amphiphilic chitosan-based polymers with (1) SH groups, (2) crosslinked with S-S between polymer chains, and (3) without SH and S-S groups were synthesized, followed by characterization by FTIR and NMR spectroscopy. Two pairs of fluorophores were investigated: 4-methylumbelliferon-trimethylammoniocinnamate-rhodamine (MUTMAC-R6G) and fluorescein isothiocyanate-rhodamine (FITC-R6G). While FITC-R6G has been described before as an FRET-producing pair, for MUTMAC-R6G, this has not been described. R6G, in addition to being an acceptor fluorophore, also serves as a model cytostatic drug in drug-release experiments. As one could expect, in aqueous solution, FRET effect was poor, but when exposed to the micelles, both MUTMAC-R6G and FITC-R6G yielded a pronounced FRET effect. Most likely, the formation of micelles is accompanied by the forced convergence of fluorophores in the hydrophobic micelle core by a donor-to-acceptor distance (r) significantly closer than in the aqueous buffer solution, which was reflected in the increase in the FRET efficiency (E). Therefore, r(E) could be used as analytical signal of the micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC), yielding values in good agreement with the literature for similar systems. We found that the r-function provides analytically valuable information about the nature and mechanism of micelle formation. S-S crosslinking between polymer chains makes the micelle more compact and stable in the normal physiological conditions, but loosens in the glutathione-rich tumor microenvironment, which is considered as an efficient approach in targeted drug delivery. Indeed, we found that R6G, as a model cytostatic agent, is released from micelles with initial rate of 5%/h in a normal tissue microenvironment, but in a tumor microenvironment model (10 mM glutathione), the release of R6G from S-S stitched polymeric micelles increased up to 24%/h. Drug-loading capacity differed substantially: from 75-80% for nonstitched polymeric micelles to ~90% for S-S stitched micelles. Therefore, appropriate FRET probes can provide comprehensive information about the micellar system, thus helping to fine-tune the drug delivery system.

Key requirements for ultraefficient sensitization in hyperfluorescence organic light-emitting diodes

Blue organic light-emitting diode (OLED) technology requires further advancements, and hyperfluorescent (HF) OLEDs have emerged as a promising solution to address stability and colour-purity concerns. A key factor influencing the performance of HF-OLEDs is Förster resonance energy transfer (FRET). Here we investigate the FRET mechanism in blue HF-OLEDs using contrasting thermally activated delayed fluorescence (TADF) sensitizers. We demonstrate that the molecular structure of the sensitizer profoundly impacts the FRET efficiency, exemplified by the spiro-linked TADF molecule ACRSA, which suppresses the dihedral-angle inhomogeneity and any lower-energy conformers that exhibit minimal FRET to the terminal emitter. Consequently, the FRET efficiency can be optimized to nearly 100%. Further, we demonstrate how the properties of a near-ideal sensitizer diverge from ideal TADF emitters. As a result, blue HF-OLEDs utilizing a greenish sensitizer exhibit a remarkable tripling of external quantum efficiency (~30%) compared with non-HF devices. This new understanding opens avenues for sensitizer design, indicating that green sensitizers can efficiently pump blue terminal emitters, thereby reducing device exciton energies and improving blue OLED stability.

A novel light-harvesting ZIF-9-TCPP as a promising FRET-based ratiometric fluorescence probe for sperm mobility.

The concentration of zinc ions in semen is significantly correlated with sperm viability and male fertility. In this work, a reliable ratiometric fluorescence probe (ZIF-9-TCPP) based on the efficient Förster resonance energy transfer (FRET) process between two luminophores, benzimidazole (BIM) and meso-tetra (4-carboxyphenyl) porphyrin (TCPP) for Zn2+ detection has been constructed, where the emissions of BIM and TCPP are used as reference and detection signals. The proximity of BIM and TCPP in one framework (ZIF-9-TCPP) and the overlapped spectra between BIM and TCPP afford the attainment of a highly efficient FRET (around 90% efficiency). Efficient FRET improves the fluorescence intensity of porphyrin to enhance the sensitivity of detection. The unique spectral shift resulting from Zn2+ binding to the porphyrin ring ensures the selectivity of detection. In addition, the response mechanism of the proposed ratiometric probes to Zn2+ has been investigated. This work provides a convenient way to design an efficient FRET system and a promising method for sperm mobility detection.

Two-photon excited-state dynamics of mEGFP-linker-mScarlet-I crowding biosensor in controlled environments.

Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Mapping the heterogeneous, dynamic crowding in living cells or tissues requires genetically encoded, site-specific, crowding sensors that are compatible with quantitative, noninvasive fluorescence micro-spectroscopy. Here, we carried out time-resolved 2P-fluorescence measurements of a new mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g L-1) via Förster resonance energy transfer (FRET) analysis. The 2P-fluorescence lifetime of the donor (mEGFP) was measured under magic-angle polarization, in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet-I), as a function of the Ficoll-70 concentration. The FRET efficiency was used to quantify the sensitivity of GE2.3 to macromolecular crowding and to determine the environmental dependence of the mEGFP-mScarlet-I distance. We also carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both macromolecular crowding and linker flexibility effects on GE2.3 rotational dynamics within the context of the Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. These time-resolved 2P-fluorescence depolarization measurements and conformational population analyses of GE2.3 were also used to estimate the free energy gain upon the structural collapse in crowded environment. Our results further the development of a rational engineering design for bioenvironmental sensors without the interference of cellular autofluorescence. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of the crowded intracellular environment under different physiological conditions.

Single-particle Förster resonance energy transfer from upconversion nanoparticles to organic dyes.

Single-particle detection and sensing, powered by Förster resonance energy transfer (FRET), offers precise monitoring of molecular interactions and environmental stimuli at a nanometric resolution. Despite its potential, the widespread use of FRET has been curtailed by the rapid photobleaching of traditional fluorophores. This study presents a robust single-particle FRET platform utilizing upconversion nanoparticles (UCNPs), which stand out for their remarkable photostability, making them superior to conventional organic donors for energy transfer-based assays. Our comprehensive research demonstrates the influence of UCNPs' size, architecture, and dye selection on the efficiency of FRET. We discovered that small particles (∼14 nm) with a Yb3+-enriched outermost shell exhibit a significant boost in FRET efficiency, a benefit not observed in larger particles (∼25 nm). 25 nm UCNPs with an inert NaLuF4 shell demonstrated a comparable level of emission enhancement via FRET as those with a Yb3+-enriched outermost shell. At the single-particle level, these FRET-enhanced UCNPs manifested an upconversion green emission intensity that was 8.3 times greater than that of their unmodified counterparts, while maintaining notable luminescence stability. Our upconversion FRET system opens up new possibilities for developing more effective high-brightness, high-sensitivity single-particle detection, and sensing modalities.

Long-persistent luminescence by host-guest Förster resonance energy transfer.

In this study, Förster resonance energy transfer (FRET) is harnessed to construct a novel stimulus-responsive long-persistent luminescence (LPL) system. Two organic molecules, DPSD and DPOD, were initially found to have no afterglow under ambient conditions, but exhibited prolonged afterglow upon friction with paper, showing a significantly promoted transition of triplet excited states. Substituting paper with α-cellulose (the main composition of paper) reveals a novel host-guest long afterglow system and allows for a deeper investigation of the above paper-promoted LPL phenomenon. The activation of the LPL effect was achieved by matrixing these components through a grinding process, capitalizing on the efficient FRET from the host to the guest owing to the appropriate energy level match, and the robust intersystem crossing (ISC) capability of the guest. This model presents a new matrix strategy to achieve efficient LPL by a facile, low cost and easy-to-handle process. Furthermore, we successfully implemented anti-counterfeiting, encryption and decryption, decoration, and water/heat stimulus-responsive applications of the obtained materials. These advancements bring LPL materials one step closer to practical commercialization.

Tailorable optical properties of polymer nanodots for triple-mode fluorescence detection of nucleic acids.

We present a triple-mode nanosensor platform for nucleic acid detection utilizing fluorescence anisotropy and Förster resonance energy transfer (FRET) strategies. The self-assembled nanoprobes serve as mass amplifiers, nanoquenchers, or nanodonors, exhibiting high FRET efficiencies (64.4-86.5%) and demonstrating excellent detection capabilities in DNA and microRNA analysis.

Heteroepitaxial Growth to Construct Hexagonal/Hexagonal β-NaYF4:Yb,Tm/Cs4PbBr6 Multi-Code Emitting Core/Shell Nanocrystals.

Synthesis of upconversion nanoparticles (UCNPs)-metal halide perovskites (MHPs) heterostructure is garnered immense attentions due to their unparalleled photophysical properties. However, the obvious difference in their structural forms makes it a huge challenge. Herein, hexagonal β-NaYF4 and hexagonal Cs4PbBr6 are filtrated to construct the UCNP/MHP heterostructural luminescent material. The similarity in their crystal structures facilitate the heteroepitaxial growth of Cs4PbBr6 on the surface of β-NaYF4 NPs, leading to the formation of high-quality β-NaYF4:Yb,Tm/Cs4PbBr6 core/shell nanocrystals (NCs). Interestingly, this heterostructure endows the core/shell NCs with typically narrow-band green emission centered at 524nm under 980nm excitation, which should be attributed to the Förster resonance energy transfer (FRET) from Tm3+ to Cs4PbBr6. It is noteworthy that the FRET efficiency of β-NaYF4:Yb,Tm/Cs4PbBr6 core/shell NCs (58.33%) is much higher than that of the physically mixed sample (1.84%). In addition, the reduced defect density, lattice anchoring effect, as well as diluted ionic bonding proportion induced by the core/shell structure further increase the excellent water-resistance and thermal cycling stability of Cs4PbBr6. These findings open up a new way to construct UCNP/MHP heterostructure with better multi-code luminescence performance and stability and promote its wide optoelectronic applications.

Im-SCC-FRET: improved single-cell-based calibration of a FRET system.

We recently developed a SCC-FRET (single-cell-based calibration of a FRET system) method to quantify spectral crosstalk correction parameters (β and δ) and system calibration parameters (G and k) of a Förster resonance energy transfer (FRET) system by imaging a single cell expressing a standard FRET plasmid with known FRET efficiency (E) and donor-acceptor concentration ratio (RC) (Liu et al., Opt. Express30, 29063 (2022)10.1364/OE.459861). Here we improved the SCC-FRET method (named as Im-SCC-FRET) to simultaneously obtain β, δ, G, k and the acceptor-to-donor extinction coefficient ratio (ε A ε D), which is a key parameter to calculate the acceptor-centric FRET efficiency (EA), of a FRET system when the range of β and δ values is set as 0-1. In Im-SCC-FRET, the target function is changed from the sum of absolute values to the sum of squares according to the least squares method, and the initial value of β and δ estimated by the integral but not the maximum value spectral overlap between fluorophore and filter. Compared with SCC-FRET, the experimental results demonstrate that Im-SCC-FRET can obtain more accurate and stable results for β, δ, G, and k, and add the ratio ε A ε D, which is necessary for the FRET hybrid assay. Im-SCC-FRET reduces the complexity of experiment preparation and opens up a promising avenue for developing an intelligent FRET correction system.

FRET-GP - A Local Measure of the Impact of Transmembrane Peptide on Lipids.

Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of proteins with lipids. In this work, we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe the nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe is quantified using generalized polarization (GP) to report on lipid mobility in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contributes to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GPFRET - the local analogue of classical GP parameter. The presence of WALP significantly increased both parameters but the local effects were considerably stronger (GPFRET ≫ GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby enhancing the depth of information obtained from the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions.

FRET-engineering of organic nanoparticles for photoacoustic imaging-guided photodynamic and augmented photothermal therapy

Phototherapy has gained a large amount of attention in tumor treatment due to its non-invasive feature. However, conventional photosensitizers suffer from the deficiency in low photon utilization efficiency, which leads to lower tumor treatment efficiency. In this contribution, a small molecular near-infrared dye BDF was synthesized to construct phototheranostic nanoparticles BDF@PEG-Ce6 NPs for combinational photodynamic therapy (PDT) and enhanced photothermal therapy (PTT). Within BDF@PEG-Ce6 NPs, Ce6 and BDF played the roles of energy donor and energy acceptor, respectively, for the Förster resonance energy transfer (FRET) process. Upon 660 nm photoirradiation, photosensitizer Ce6 within BDF@PEG-Ce6 NPs not only demonstrated photodynamic activity but also served as an energy generator to release energy (donor) to be absorbed by BDF dye with FRET efficiency of 97.7%. Thus, BDF@PEG-Ce6 NPs showed an outstanding photothermal effect, enabling photoacoustic (PA) imaging of tumor sites to guide tumor treatment. It is believed that this work can provide a new idea for the rational design of photosensitizers for better tumor treatment in the future.

Giant Light‐Harvesting in Dye‐Loaded Nanoparticles Enhanced by Blank Hydrophobic Salts

AbstractLight‐harvesting is a fundamental process in nature, which inspires researchers to develop artificial systems for photocatalysis, photovoltaics, and biosensing. A previously introduced light‐harvesting nanoantenna, based on polymeric nanoparticles (NPs) loaded with rhodamine dyes and bulky hydrophobic counterions, provides a record‐breaking antenna effect ≈1000. However, the high dye cooperativity of its thousands of encapsulated dyes causes energy losses by traces of self‐quenched dye aggregates. Here, it is found that these imperfections can be suppressed by blank hydrophobic salts (BHS) formed by the same bulky counterion (fluorinated tetraphenylborate) with an optically inactive cation, analogs of ionic liquids. The presence of BHS increases twofold the fluorescence quantum yields and fluorescence lifetimes of NPs and suppresses their fluorescence blinking. This study assumes that BHS provides an excess of bulky counterions that excludes traces of dye aggregates. As a result, an efficient Forster resonance energy transfer (FRET) is achieved from 40 000 dye donors to a single acceptor within a 70 nm particle, leading to the antenna effect of 4800, which is by far the highest value reported to date. Using this nanoantenna, a single‐molecule detection of the FRET acceptor is realized at low excitation power using an RGB camera of a smartphone.

Resonance energy transfer in electrostatically assembled donor-acceptor system based on blue-emitting InP quantum dots

Demonstration of light harvesting studies with blue-emitting quantum dots (QDs) is important to expand the scope of QDs in various optoelectronic applications including display, photocatalysis, energy and electron transfer processes, sensing, etc. Here, we report a light induced Förster resonance energy transfer (FRET) process in an all-QD based nanohybrid system containing pure-blue emitting indium phosphide (InP) QDs. A precise control over the kinetics of nucleation and growth processes resulted in the formation of environmentally friendly blue and green emitting InP QDs. The surfaces of the QDs were functionalized with oppositely charged ligands to form an electrostatically assembled donor-acceptor system based on blue and green emitting InP QDs. A large spectral overlap integral and the close proximity between the QDs in the electrostatically assembled donor-acceptor system led to an efficient FRET process (∼65%) from blue-emitting InP QDs to green-emitting InP QDs in water. Systematic steady-state and time-resolved spectroscopic studies revealed the involvement of both static as well as dynamic components in the photoluminescence quenching process. Control experiments conclusively prove the necessity of appropriately functionalized QDs and electrostatic attraction for achieving an efficient FRET process. In short, our work proves the ability of blue-emitting InP QDs to participate in an efficient FRET process, which can accelerate the development of all-QD based light harvesting systems for various optoelectronic applications.

Incoherent Energy Transfer between the Baseplate and FMO Protein Explored at Ideal Geometries.

The Förster resonance energy transfer (FRET) between the Fenna-Matthews-Olson (FMO) protein complex and the chlorosomal baseplate (CBP) is investigated by using an idealized model. This simplified model is based on crystal structure and molecular dynamics conformations. Some of the further input, such as the transition dipole moments, was extracted from earlier molecular-level simulations. The resulting model mimics the effects of the relative position between the CBP and the FMO complex on the corresponding FRET efficiency under ideal conditions, involving about 1.3 billion FRET calculations per investigated model. In this idealized model and employing some approximations, it is found that FRET efficiency is almost completely independent of the FMO trimer orientation (displacement, distance, and rotation), despite FMO and CBP being highly structured complexes. Even removing individual FMO BChl triples will only reduce the FRET efficiency by up to 8.6%. An FMO containing only the least efficient BChl triple will retain about 25% of the FRET efficiency of a full FMO complex. In addition to its proposed function as an energetic funnel, FMO is thus identified to act as a highly robust spatial funnel for CBP excitation harvesting, independent of the mutual CBP-FMO orientation.

LuF3: Yb3+/Tm3+@Lu6O5F8: Yb3+/Tm3+@BiOI for efficient degradation of pollutants under NIR light: Synergy of enhanced upconversion emission and efficient FRET

Effective use of near-infrared (NIR) light for photocatalysis in the solar full-spectrum remains a major challenge. Herein, LuF3: Yb3+/Tm3+@Lu6O5F8: Yb3+/Tm3+ (LYT@LOYT), which could convert NIR light into UV–visible light, was combined with BiOI to form a novel core-shell structured LuF3: Yb3+/Tm3+@Lu6O5F8: Yb3+/Tm3+@BiOI (LYT@LOYT@BiOI) upconversion photocatalyst. LOYT shell layer was grown in situ on LYT by high-temperature calcination in air, resulting in a 2.36-fold enhancement of blue light emission from the upconversion luminescent core. The tight core-shell wrapping formed by the solvothermal method achieved an efficient Förster resonance energy transfer (FRET) between LYT@LOYT and BiOI. Meanwhile, the formation of heterogeneous structure also accelerated the separation and transfer of photogenerated electron-hole pairs. As a result, under NIR and Vis-NIR irradiation, the degradation rates of LYT@LOYT(20)@BiOI for bisphenol A (BPA) were 1.24 and 1.11 times of LYT(20)@BiOI, and 1.57 and 1.30 times of BiOI, respectively. The enhanced photocatalytic activity was attributed to the expanded photoresponse range, enhanced upconversion emission, efficient energy transfer process, and excellent electron-hole pairs separation transfer efficiency. h+ and ⋅O2- were shown to be active species in the degradation process. This work provides a useful reference for the rational design of solar full-spectrum responsive photocatalysts which can efficiently utilize NIR light.

Single probe-based catalytic quantum dot FRET nanosensor for human alkyladenine DNA glycosylase detection

Human alkyladenine DNA glycosylase (hAAG) is essential for repairing alkylated and deaminated bases, and it has become a prospective diagnosis biomarker and a therapeutic target for disease treatment. However, most of hAAG assays suffer from complicated reaction scheme, poor specificity, long assay time, and limited sensitivity. Herein, we report a novel single probe-based catalytic quantum dot (QD) Förster resonance energy transfer (FRET) nanosensor for simple and sensitive detection of hAAG activity. In this assay, hAAG induces the generation of 3′ OH terminus via the excision of I base and the cleavage of AP site by APE1, subsequently initiating strand displacement reaction to produce numerous ssDNA signal probes. These probes can self-assemble on the QD surface to induce efficient FRET between QD and Cy5. This assay is very simple with the involvement of only a single probe for the achievement of both specific sensing and efficient signal amplification. Moreover, each signal probe contains multiple Cy5 moieties, and multiple signal probes can assemble on a single QD to greatly enhance the FRET efficiency. This nanosensor exhibits a detection limit of 3.60 × 10−10 U/μL and it is suitable for measuring enzymatic kinetics, screening inhibitor, and quantifying cellular hAAG activity with single-cell sensitivity.

Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

Fluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single-molecule or single-particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during Förster resonance energy transfer (FRET) analysis in the presence of a significant donor-only fraction. In this article we investigate whether an object localization strategy and the phasor approach to FLIM have beneficial effects when carrying out FRET analyses of single particles. Using simulations, we first showed that an average of ∼300 photons, spread over the different pixels encompassing single fluorescing particles and without background, is enough to determine a correct phasor signature (SD < 5% for a 4-ns lifetime). For immobilized single- or double-labeled dsDNA molecules, we next validated that particle-based phasor-FLIM-FRET readily allows estimating fluorescence lifetimes and FRET from single molecules. Thirdly, we applied particle-based phasor-FLIM-FRET to investigate protein-protein interactions in subdiffraction HIV-1 viral particles. To do this, we first quantitatively compared the fluorescence brightness, lifetime, and photostability of different popular fluorescent protein-based FRET probes when genetically fused to the HIV-1 integrase enzyme in viral particles, and conclude that eGFP, mTurquoise2, and mScarlet perform best. Finally, for viral particles coexpressing FRET-donor/acceptor-labeled IN, we determined the absolute FRET efficiency of IN oligomers. Available in a convenient open-source graphical user interface, we believe that particle-based phasor-FLIM-FRET is a promising tool to provide detailed insights in samples suffering from low overall signal intensities.

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.

Influence of the Fluorophore Mobility on Distance Measurements by Gas-Phase FRET.

Gas-phase Förster resonance energy transfer (FRET) combines mass spectrometry and fluorescence spectroscopy for the conformational analysis of mass-selected biomolecular ions. In FRET, fluorophore pairs are typically covalently attached to a biomolecule using short linkers, which affect the mobility of the dye and the relative orientation of the transition dipole moments of the donor and acceptor. Intramolecular interactions may further influence the range of motion. Yet, little is known about this factor, despite the importance of intramolecular interactions in the absence of a solvent. In this study, we applied transition metal ion FRET (tmFRET) to probe the mobility of a single chromophore pair (Rhodamine 110 and Cu2+) as a function of linker lengths to assess the relevance of intramolecular interactions. Increasing FRET efficiencies were observed with increasing linker length, ranging from 5% (2 atoms) to 28% (13 atoms). To rationalize this trend, we profiled the conformational landscape of each model system using molecular dynamics (MD) simulations. We captured intramolecular interactions that promote a population shift toward smaller donor-acceptor separation for longer linker lengths and induce a significant increase in the acceptor's transition dipole moment. The presented methodology is a first step toward the explicit consideration of a fluorophore's range of motion in the interpretation of gas-phase FRET experiments.

Single-component organic semiconducting polymer nanoparticles for near-infrared afterglow imaging

Afterglow imaging holds great promise in the field of bioimaging owing to its ultrahigh imaging sensitivity. However, most reported afterglow imaging nanoprobes are prepared via nanoprecipitation, which both their stability and Förster resonance energy transfer (FRET) efficiency can further be improved. Herein, we report a single-component organic afterglow nanoprobe (PPVP-PEG) for lymph nodes and tumor imaging. PPVP-PEG is synthesized by conjugating both near-infrared (NIR) emitting photosensitizer (tetraphenylporphyrin, TPP) and poly(ethylene glycol) (PEG) onto side chains of polyphenylenevinylene (PPV). Such structure makes PPVP-PEG an amphiphilic copolymer that can self-assemble into water without the addition of stabilizer. PPVP-PEG has NIR afterglow emission and higher afterglow intensity than PPV-PEG without TPP conjugation. In addition, PPVP-PEG has a higher FRET efficiency between TPP and PPV than similar nanoprobe prepared via nanoprecipitation. The afterglow signal of PPVP-PEG can clearly detect lymph nodes of mice with high sensitivity. In addition, owing to the unique structure, PPVP-PEG shows excellent tumor targeting capability. Combining with the high sensitivity of afterglow imaging, the tumor can be imaged with high signal-to-background ratio. Thus, this work provides an approach to improve the stability and FRET efficiency of afterglow probe and may boost the development of afterglow imaging.