PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends.

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions.

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.

Enhanced Solid-State Fluorescence of Flavin Derivatives by Incorporation in the Metal-Organic Frameworks MIL-53(Al) and MOF-5.

The flavin derivatives 10-methyl-isoalloxazine (MIA) and 6-fluoro-10-methyl-isoalloxazine (6F-MIA) were incorporated in two alternative metal-organic frameworks, (MOFs) MIL-53(Al) and MOF-5. We used a post-synthetic, diffusion-based incorporation into microcrystalline MIL-53 powders with one-dimensional (1D) pores and an in-situ approach during the synthesis of MOF-5 with its 3D channel network. The maximum amount of flavin dye incorporation is 3.9 wt% for MIA@MIL-53(Al) and 1.5 wt% for 6F-MIA@MIL-53(Al), 0.85 wt% for MIA@MOF-5 and 5.2 wt% for 6F-MIA@MOF-5. For the high incorporation yields the probability to have more than one dye molecule in a pore volume is significant. As compared to the flavins in solution, the fluorescence spectrum of these flavin@MOF composites is broadened at the bathocromic side especially for MIA. Time-resolved spectroscopy showed that multi-exponential fluorescence lifetimes were needed to describe the decays. The fluorescence-weighted lifetime of flavin@MOF of 4 ± 1 ns also corresponds to those in solution but is significantly prolonged compared to the solid flavin dyes with less than 1 ns, thereby confirming the concept of "solid solutions" for dye@MOF composites. The fluorescence quantum yield (ΦF) of the flavin@MOF composites is about half of the solution but is significantly higher compared to the solid flavin dyes. Both the fluorescence lifetime and quantum yield of flavin@MOF decrease with the flavin loading in MIL-53 due to the formation of various J-aggregates. Theoretical calculations using plane-wave and QM/MM methods are in good correspondence with the experimental results and explain the electronic structures as well as the photophysical properties of crystalline MIA and the flavin@MOF composites. In the solid flavins, π-stacking interactions of the molecules lead to a charge transfer state with low oscillator strength resulting in aggregation-caused quenching (ACQ) with low lifetimes and quantum yields. In the MOF pores, single flavin molecules represent a major population and the computed MIA@MOF structures do not find π-stacking interactions with the pore walls but only weak van-der-Waals contacts which reasons the enhanced fluorescence lifetime and quantum yield of the flavins in the composites compared to their neat solid state. To analyze the orientation of flavins in MOFs, we measured fluorescence anisotropy images of single flavin@MOF-5 crystals and a static ensemble flavin@MIL53 microcrystals, respectively. Based on image information, anisotropy distributions and overall curve of the time-resolved anisotropy curves combined with theoretical calculations, we can prove that all fluorescent flavins species have a defined and rather homogeneous orientation in the MOF framework. In MIL-53, the transition dipole moments of flavins are orientated along the 1D channel axis, whereas in MOF-5 we resolved an average orientation that is tilted with respect to the cubic crystal lattice. Notably, the more hydrophobic 6F-MIA exhibits a higher degree order than MIA. The flexible MOF MIL-53(Al) was optimized essentially to the experimental large-pore form in the guest-free state with QuantumEspresso (QE) and with MIA molecules in the pores the structure contracted to close to the experimental narrow-pore form which was also confirmed by PXRD. In summary, the incorporation of flavins in MOFs yields solid-state materials with enhanced rigidity, stabilized conformation, defined orientation and reduced aggregations of the flavins, leading to increased fluorescence lifetime and quantum yield as controllable photo-luminescent and photo-physical properties.

Pyrolysis and Solvothermal Synthesis for Carbon Dots: Role of Purification and Molecular Fluorophores.

Over the last decade, the interest in carbon dots, graphene dots, or similar carbon-based nanoparticles has increased considerably. This interest is based on potentially high fluorescent quantum yields, controllable excitation-dependent emission, low toxicity, and convenient reaction conditions. Carbon dots are often seen as a promising alternative to classical semiconductor quantum dots that are typically made from toxic semiconductor materials. Surprisingly, aspects like the atomic structure, composition, mechanism of formation, and precise understanding of the photophysical properties of carbon dots are still mostly unknown. The large number of different precursor systems and the variety in synthesis routes make a direct comparison of different systems difficult. To advance this, we went for a systematic approach and compared the results of four synthesis routes using two different precursor systems. We used different spectroscopy and microscopy methods including fluorescence correlation spectroscopy to characterize the different reaction products. We found that for syntheses solely based on citric acid as the precursor, we obtain particles where the emission wavelength is strongly dependent on the excitation wavelength despite relatively low quantum yields. In comparison, when urea is added as a nitrogen doping reactant, we observe vastly increased quantum yields. By making use of a combination of dialysis and column chromatography, we were able to isolate various luminescent species with high quantum yields and verify the existence of different molecular fluorophores. A detailed and consistent characterization of the reaction products during the course of purification revealed strong interactions between molecular fluorophores and larger reaction products.

Dynamics of the nucleosomal histone H3 N-terminal tail revealed by high precision single-molecule FRET.

Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the µs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Excited state dipole moments and lifetimes of 2-cyanoindole from rotationally resolved electronic Stark spectroscopy.

The permanent dipole moments of 2-cyanoindole (cyanoindole = CNI) in its ground and lowest excited singlet states have been determined from rotationally resolved electronic Stark spectroscopy under jet-cooled conditions. From the orientation of the transition dipole moment and the geometry changes upon electronic excitation the lowest excited singlet state could be shown to be of Lb-symmetry. The general statement, that the La-state has the larger permanent dipole moment of the two lowest excited singlet states, will be challenged in this contribution. On the basis of the different electronic nature of the first excited singlet state the behavior of 2-, 3-, 4- and 5-CNI is discussed. The excited state lifetime of isolated 2-CNI in the gas phase has been determined to be 9.4 ns. This value is compared to the excited state lifetime in ethyl acetate solution of 2.6 ns, which was quantified with a Strickler-Berg analysis. Using water as solvent shortens the 2-CNI lifetime to <40 ps. The reason for this drastic shortening is discussed in detail. Additionally, the rotationally resolved electronic spectrum of 2-CNI(1-d1) has been measured and analyzed.

On the large apparent Stokes shift of phthalimides.

The photophysics of N-methylphthalimide (MP) in solution (cyclohexane, ethanol, acetonitrile, and water) was characterized by steady state as well as time resolved fluorescence and absorption spectroscopy. In all solvents the compound exhibits an unusually large Stokes shift of ∼10 000 cm-1. It is attributed to an ultrafast (<100 fs) depletion of the initially excited state, which results in the population of a weakly emitting state. Quantum chemical computations (DFT-MRCI) support this. They identify two energetically low-lying singlet ππ* excitations of different oscillator strength. Whereas the Stokes shift and thereby the ultrafast depletion of the initial excitation are hardly affected by the solvent later processes respond strongly. The fluorescence lifetime varies from ∼10 ps (cyclohexane) to ∼3 ns (water). This is attributed to a varying energetic accessibility of nπ* excitations.

Rotationally resolved electronic spectroscopy of 3-cyanoindole and the 3-cyanoindole-water complex.

The rotationally resolved electronic spectra of the origin bands of 3-cyanoindole, 3-cyanoindole(d1), and the 3-cyanoindole-(H2O)1 cluster have been measured and analyzed using evolutionary algorithms. For the monomer, permanent dipole moments of 5.90 D for the ground state, and of 5.35 D for the lowest excited singlet state have been obtained from electronic Stark spectroscopy. The orientation of the transition dipole moment is that of an 1Lb state for the monomer. The water moiety in the water cluster could be determined to be trans-linearly bound to the NH group of 3-cyanoindole, with an NHO hydrogen bond length of 201.9 pm in the electronic ground state. Like the 3-cyanoindole monomer, the 3-cyanoindole-water cluster also shows an 1Lb-like excited singlet state. The excited state lifetime of isolate 3-cyanoindole in the gas phase has been determined to be 9.8 ns, and that of 3-cyanoindole(d1) has been found to be 14.8 ns, while that of the 1 : 1 water cluster is considerably shorter (3.6 ns). The excited state lifetime of 3-cyanoindole(d1) in D2O solution has been found to be smaller than 20 ps.

Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved.

Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from single-molecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.

An optofluidic antenna for enhancing the sensitivity of single-emitter measurements.

Many single-molecule investigations are performed in fluidic environments, for example, to avoid unwanted consequences of contact with surfaces. Diffusion of molecules in this arrangement limits the observation time and the number of collected photons, thus, compromising studies of processes with fast or slow dynamics. Here, we introduce a planar optofluidic antenna (OFA), which enhances the fluorescence signal from molecules by about 5 times per passage, leads to about 7-fold more frequent returns to the observation volume, and significantly lengthens the diffusion time within one passage. We use single-molecule multi-parameter fluorescence detection (sm-MFD), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) measurements to characterize our OFAs. The antenna advantages are showcased by examining both the slow (ms) and fast (50 µs) dynamics of DNA four-way (Holliday) junctions with real-time resolution. The FRET trajectories provide evidence for the absence of an intermediate conformational state and introduce an upper bound for its lifetime. The ease of implementation and compatibility with various microscopy modalities make OFAs broadly applicable to a diverse range of studies.

Resolving dynamics and function of transient states in single enzyme molecules.

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.

Fluorophores as optical sensors for local forces.

The main aim of this study is to investigate correlations between the impact of an external mechanical force on the molecular framework of fluorophores and the resultant changes in their fluorescence properties. Taking into account previous theoretical studies, we designed a suitable custom-tailored oligoparaphenylenevinylene derivative (OPV5) with a twisted molecular backbone. Thin foils made of PVC doped with 100 nM OPV were prepared. By applying uniaxial force, the foils were stretched and three major optical effects were observed simultaneously. First, the fluorescence anisotropy increased, which indicates a reorientation of the fluorophores within the matrix. Second, the fluorescence lifetime decreased by approximately 2.5% (25 ps). Finally, we observed an increase in the emission energy of about 0.2% (corresponding to a blue-shift of 1.2 nm). In addition, analogous measurements with Rhodamine 123 as an inert reference dye showed only minor effects, which can be attributed to matrix effects due to refractive index changes. To relate the observed spectroscopic changes to the underlying changes in molecular properties, quantum-chemical calculations were also performed. Semiempirical methods had to be used because of the size of the OPV5 chromophore. Two conformers of OPV5 (C(2) and C(i) symmetry) were considered and both gave very similar results. Both the observed blue-shift of fluorescence and the reduced lifetime of OPV5 under tensile stress are consistent with the results of the semiempirical calculations. Our study proves the feasibility of fluorescence-based local force probes for polymers under tension. Improved optical sensors of this type should in principle be able to monitor local mechanical stress in transparent samples down to the single-molecule level, which harbors promising applications in polymer science and nanotechnology.

Integrated NMR, Fluorescence, and Molecular Dynamics Benchmark Study of Protein Mechanics and Hydrodynamics.

Understanding the function of a protein requires not only knowledge of its tertiary structure but also an understanding of its conformational dynamics. Nuclear magnetic resonance (NMR) spectroscopy, polarization-resolved fluorescence spectroscopy and molecular dynamics (MD) simulations are powerful methods to provide detailed insight into protein dynamics on multiple time scales by monitoring global rotational diffusion and local flexibility (order parameters) that are sensitive to inter- and intramolecular interactions, respectively. We present an integrated approach where data from these techniques are analyzed and interpreted within a joint theoretical description of depolarization and diffusion, demonstrating their conceptual similarities. This integrated approach is then applied to the autophagy-related protein GABARAP in its cytosolic form, elucidating its dynamics on the pico- to nanosecond time scale and its rotational and translational diffusion for protein concentrations spanning 9 orders of magnitude. We compare the dynamics of GABARAP as monitored by 15N spin relaxation of the backbone amide groups, fluorescence anisotropy decays and fluorescence correlation spectroscopy of side chains labeled with BODIPY FL, and molecular movies of the protein from MD simulations. The recovered parameters agree very well between the distinct techniques if the different measurement conditions (probe localization, sample concentration) are taken into account. Moreover, we propose a method that compares the order parameters of the backbone and side chains to identify potential hinges for large-scale, functionally relevant intradomain motions, such as residues 27/28 at the interface between the two subdomains of GABARAP. In conclusion, the integrated concept of cross-fertilizing techniques presented here is fundamental to obtaining a comprehensive quantitative picture of multiscale protein dynamics and solvation. The possibility to employ these validated techniques under cellular conditions and combine them with fluorescence imaging opens up the perspective of studying the functional dynamics of GABARAP or other proteins in live cells.

Impact of Mono-Fluorination on the Photophysics of the Flavin Chromophore.

Three mono-fluorinated derivatives of the flavin core system 10-methyl-isoalloxazine (MIA) were synthesized. Aqueous solutions of these compounds were characterized by steady-state and time-resolved spectroscopy. The positions for the fluorination (6, 7 and 8) were motivated by the nodal structure of the frontier orbitals of MIA. In comparison with MIA, the fluorination results in bathochromic (6F- and 7F-MIA) and hypsochromic (8F-MIA) shifts of the adiabatic excitation energy of the lowest allowed transition. Shifts of up to ~500 cm-1 were observed. These spectroscopic shifts go along with changes in fluorescence quantum yields and lifetimes. In addition, triplet yields are affected. For 7F-MIA, a 50% increase in the fluorescence quantum yield as well as a 50% decrease in triplet yield is observed rendering the compound interesting for fluorescence applications. The measured effects are discussed in terms of qualitative perturbation theory.

High precision FRET studies reveal reversible transitions in nucleosomes between microseconds and minutes.

Nucleosomes play a dual role in compacting the genome and regulating the access to DNA. To unravel the underlying mechanism, we study fluorescently labeled mononucleosomes by multi-parameter FRET measurements and characterize their structural and dynamic heterogeneity upon NaCl-induced destabilization. Species-selective fluorescence lifetime analysis and dynamic photon distribution analysis reveal intermediates during nucleosome opening and lead to a coherent structural and kinetic model. In dynamic octasomes and hexasomes the interface between the H2A-H2B dimers and the (H3-H4)2 tetramer opens asymmetrically by an angle of ≈20° on a 50 and 15 µs time scale, respectively. This is followed by a slower stepwise release of the dimers coupled with DNA unwrapping. A mutation (H2A-R81A) at the interface between H2A and H3 facilitates initial opening, confirming the central role of the dimer:tetramer interface for nucleosome stability. Partially opened states such as those described here might serve as convenient nucleation sites for DNA-recognizing proteins.

Fluorescence detection with high time resolution: from optical microscopy to simultaneous force and fluorescence spectroscopy.

Picosecond time-resolution fluorescence signal detection over many hours is possible using the time-correlated single photon counting (TCSPC) technique. Advanced TCSPC with clock oscillator set by the pulsed laser and data analysis provides a tool to investigate processes in single molecules on time scale from picoseconds to seconds. Optical imaging techniques combined with TCSPC allow one to study the spatial distribution of fluorescence properties in solution and on a surface. Mechanical manipulation of a single macromolecule by means of an atomic-force microscope makes it possible to detect fluorescence signal changes as a function of mechanical conformations of a fluorescent dye attached to a single DNA molecule.

Guanylate binding proteins directly attack Toxoplasma gondii via supramolecular complexes.

GBPs are essential for immunity against intracellular pathogens, especially for Toxoplasma gondii control. Here, the molecular interactions of murine GBPs (mGBP1/2/3/5/6), homo- and hetero-multimerization properties of mGBP2 and its function in parasite killing were investigated by mutational, Multiparameter Fluorescence Image Spectroscopy, and live cell microscopy methodologies. Control of T. gondii replication by mGBP2 requires GTP hydrolysis and isoprenylation thus, enabling reversible oligomerization in vesicle-like structures. mGBP2 undergoes structural transitions between monomeric, dimeric and oligomeric states visualized by quantitative FRET analysis. mGBPs reside in at least two discrete subcellular reservoirs and attack the parasitophorous vacuole membrane (PVM) as orchestrated, supramolecular complexes forming large, densely packed multimers comprising up to several thousand monomers. This dramatic mGBP enrichment results in the loss of PVM integrity, followed by a direct assault of mGBP2 upon the plasma membrane of the parasite. These discoveries provide vital dynamic and molecular perceptions into cell-autonomous immunity.

Analyzing Förster resonance energy transfer with fluctuation algorithms.

Fluorescence correlation spectroscopy (FCS) in combination with Förster resonance energy transfer (FRET) has been developed to a powerful statistical tool, which allows for the analysis of FRET fluctuations in the huge time of nanoseconds to seconds. FRET-FCS utilizes the strong distance dependence of the FRET efficiency on the donor (D)-acceptor (A) distance so that it developed to a perfect method for studying structural fluctuation in biomolecules involved in conformational flexibility, structural dynamics, complex formation, folding, and catalysis. Structural fluctuations thereby result in anticorrelated donor and acceptor signals, which are analyzed by FRET-FCS in order to characterize underlying structural dynamics. Simulated and experimental examples are discussed. First, we review experimental implementations of FRET-FCS and present theory for a two-state interconverting system. Additionally, we consider a very common case of FRET dynamics in the presence of donor-only labeled species. We demonstrate that the mean relaxation time for the structural dynamics can be easily obtained in most of cases, whereas extracting meaningful information from correlation amplitudes can be challenging. We present a strategy to avoid a fit with an underdetermined model function by restraining the D and A brightnesses of the at least one involved state, so that both FRET efficiencies and both rate constants (i.e., the equilibrium constant) can be determined. For samples containing several fluorescent species, the use of pulsed polarized excitation with multiparameter fluorescence detection allows for filtered FCS (fFCS), where species-specific correlation functions can be obtained, which can be directly interpreted. The species selection is achieved by filtering using fluorescence decays of individual species. Analytical functions for species auto- and cross-correlation functions are given. Moreover, fFCS is less affected by photophysical artifacts and often offers higher contrast, which effectively increases its time resolution and significantly enhances its capability to resolve multistate kinetics. fFCS can also differentiate between species even when their brightnesses are the same and thus opens up new possibilities to characterize complex dynamics. Alternative fluctuation algorithms to study FRET dynamics are also briefly reviewed.