Understanding how supramolecular structures grow requires imaging methods that combine nanometer-scale resolution with real-time operation under native conditions, yet super-resolution microscopy typically relies on extrinsic dyes that may perturb assembly pathways. Here, we show that supramolecular fibers from a saccharide-derived benzaldehyde (SachCHO) exhibit both Solid-state Luminescence Enhancement (SLE) and Solid-state Luminescence Intermittency (SLI), enabling label-free stochastic optical reconstruction microscopy (STORM). Crucially, the SLI is not imparted by external labelling but is fundamentally linked to the assembly process itself, localizing at structural defects within the fibers via a defect-mediated redox-blinking mechanism. Using SLI-STORM, we visualize fiber self-assembly with ~25 nm resolution and identify these defects as hotspots of heterogeneous growth, revealing intermittent elongation and non-classical kinetic growth regimes. Analogous behavior in amyloid fibrils suggests this phenomenon is general. By leveraging the material's own photo-physics/chemistry, SLI-STORM transforms the assembling structure into its own dynamic reporter, offering a minimally invasive strategy to probe growth and defect formation in both synthetic and biological supramolecular materials.

Singlet fission (SF) is a photophysical process where a singlet excitation generates two triplet excited states, enhancing exciton multiplication potentially useful for solar energy conversion. Since SF typically outcompetes radiative decay, single molecule studies of SF have remained elusive. Here, we present single molecule spectroscopy of a terrylenediimide (TDI) dimer at room and cryogenic temperatures. By analyzing the stream of photons emitted by single dimers, the rates of formation and decay of SF-born triplet states were determined. We report considerable static and dynamic heterogeneities of the SF process, which are reflected in broad rate distributions as well as the occasional occurrence of delayed fluorescence and rate fluctuations during spin evolution. Cryogenic experiments point to the formation of a coherent multiexciton superposition state that decays into the singlet exciton from which a correlated triplet pair evolves. Our results establish single molecule spectroscopy as a new avenue into mechanistic details of the SF process which often are drowned by ensemble averaging.

We perform laser spectroscopy at liquid helium temperatures (T = 2 K) to investigate single dibenzoterrylene (DBT) molecules doped in anthracene crystals of nanoscopic height fabricated by electrohydrodynamic dripping. Using high-resolution fluorescence excitation spectroscopy, we show that zero-phonon lines of single molecules in printed nanocrystals are nearly as narrow as the Fourier-limited transitions observed for the same guest-host system in the bulk. Moreover, the spectral instabilities are comparable to or less than one line width. By recording super-resolution images of DBT molecules and varying the polarization of the excitation beam, we determine the dimensions of the printed crystals and the orientation of the crystals' axes. Electrohydrodynamic printing of organic nano- and microcrystals is of interest for a series of applications, where controlled positioning of quantum emitters with narrow optical transitions is desirable.

The fluorescence quantum yield of organic NIR-emitters is typically limited by internal conversion (IC), restricting their applications in imaging and quantum technology. Here, we study the impact of deuteration and temperature on the emission properties of dibenzoterrylene (DBT) by bulk and single molecule spectroscopy. Based on simple photophysical modelling, we first clarify how IC affects the single molecule emission rate. Next, we show that deuteration of DBT leads to a concomitant increase in the fluorescence lifetime and quantum yield by up to 60%. This clear deuterium isotope effect indicates a significant contribution of C-H-vibrations in the IC process. The solvent-dependent changes in the IC rate of hydrogenated and deuterated compounds were found to follow the predictions of the energy gap law in the weak coupling limit. This view is supported by the very weak temperature dependence of the IC rate between 5 and 300 K. Our results not only shed light on the non-radiative relaxation of a topical polycyclic aromatic hydrocarbon, but also pave the way for single molecule quantum emitters with high emission yields in the NIR.

Organic near-infrared (NIR) photoblinking fluorophores are highly desirable for live-cell super-resolution imaging based on single-molecule localization microscopy (SMLM). Herein we introduce a novel small chromophore, PMIP, through the fusion of perylenecarboximide with 2,2-dimetheylpyrimidine. PMIP exhibits an emission maximum at 732 nm with a high fluorescence quantum yield of 60% in the wavelength range of 700-1000 nm and excellent photoblinking without any additives. With resorcinol-functionalized PMIP (PMIP-OH), NIR SMLM imaging of lysosomes is demonstrated for the first time in living mammalian cells under physiological conditions. Moreover, metabolically labeled nascent DNA is site-specifically detected using azido-functionalized PMIP (PMIP-N-3 ) via click chemistry, thereby enabling the super-resolution imaging of nascent DNA in phosphate-buffered saline with a 9-fold improvement in spatial resolution. These results indicate the potential of PMIP-based NIR blinking fluorophores for biological applications of SMLM.

Ligand-protein binding assays based on intrinsic protein fluorescence are straightforward, inexpensive methods to study ligand-protein interactions. However, their applicability is limited to ligands that can interfere with protein emission. In this Note, we describe the applicability of 2,2'-bithiophene as a FRET-based sensor tag, that can be incorporated into high-affinity ligands to generate target-specific compounds able to quench protein fluorescence upon binding. The generated ligands were assessed in different assay designs. Considerations to account for possible sources of interference with the assay readout are addressed, besides interpretation of the obtained results.

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The KD value observed is in accordance with the IC50 determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.

Zika and dengue viruses cause mosquito-borne diseases of high epidemic relevance. The viral NS2B-NS3 proteases play crucial roles in the pathogen replication cycle and are validated drug targets. They can adopt at least two conformations depending on the position of the NS2B cofactor. Recently, we reported ligand-induced conformational changes of dengue virus NS2B-NS3 protease by single-molecule Förster resonance energy transfer (smFRET). Here, we investigated the conformational dynamics of the homologous Zika virus protease through an integrated methodological approach combining smFRET, thermal shift assays (DSF and nanoDSF) and 19F NMR spectroscopy. Our results show that allosteric inhibitors favor the open conformation and competitive inhibitors stabilize the closed conformation of the Zika virus protease.

Abstract Protein‐ligand interactions are crucial for many cellular processes, with details of the binding mechanism being discussed as essential for biological functions. Interestingly, protein binding often involves conformational changes between two or more states, whereby different binding mechanisms are possible even with a simple two‐state description. Two models are widely used to portray protein‐ligand interactions: Induced fit and conformational selection. However, distinguishing them experimentally is challenging. Single‐molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool to resolve structural dynamics at the level of single proteins. Here, we investigated immobilized Zika virus (ZIKV) and dengue virus (DENV2) NS2B‐NS3 proteases using smFRET to compare their conformational changes upon binding to competitive small molecule inhibitors. The analysis of the smFRET data allowed us to distinguish between induced fit and conformational selection models and assign the binding mechanism from the kinetic parameters obtained. Although DENV and ZIKV protease are proteins with high structural similarities, our results reveal that they have opposite binding mechanisms for competitive ligands. While the protein‐ligand interaction in the ZIKV protease follows an induced fit mechanism, the DENV protease follows the conformational selection mechanism.

A terrylenedicarboximide-anthraquinone dyad, FTQ, with absorption in the second near-infrared region (NIR-II) is obtained as a high-performance chromophore for photothermal therapy (PTT). The synthetic route proceeds by C-N coupling of amino-substituted terrylenedicarboximide (TMI) and 1,4-dichloroanthraquinone followed by alkaline-promoted dehydrocyclization. FTQ with extended pi-conjugation exhibits an optical absorption band peaking at 1140 nm and extending into the 1500 nm range. Moreover, as determined by dielectric spectroscopy in dilute solutions, FTQ achieves an ultrastrong dipole moment of 14.4 +/- 0.4 Debye due to intense intramolecular charge transfer. After encapsulation in a biodegradable polyethylene glycol (DSPE-mPEG2000), FTQ nanoparticles (NPs) deliver a high photothermal conversion efficiency of 49% under 1064 nm laser irradiation combined with excellent biocompatibility, photostability, and photoacoustic imaging capability. In vitro and in vivo studies reveal the great potential of FTQ NPs in photoacoustic-imaging-guided photothermal therapy for orthotopic liver cancer treatment in the NIR-II window.

A new class of near-infrared (NIR) fluorophores, PAI , is obtained by consecutive C−N/C−C bond formation between diphenylamines and 9,10-dibromoperylenecarboximide. Owing to the rigid structure, extended π-conjugation and pronounced push-pull substitution, these fluorophores show emission maxima up to 804 nm and large Stokes shifts. The extraordinarily high fluorescence quantum yields from 47 % to 70 % are attributed to chloro substitution in the bay positions of the perylene core. These characteristics, together with high photostability, qualify them as useful NIR emitters for applications as biomarkers and security inks.

The photoluminescence (PL) of metal halide perovskites can recover after light or current-induced degradation. This self-healing ability is tested by acting mechanically on MAPbI3 polycrystalline microcrystals by an atomic force microscope tip (applying force, scratching, and cutting) while monitoring the PL. Although strain and crystal damage induce strong PL quenching, the initial balance between radiative and nonradiative processes in the microcrystals is restored within a few minutes. The stepwise quenching-recovery cycles induced by the mechanical action is interpreted as a modulation of the PL blinking behavior. This study proposes that the dynamic equilibrium between active and inactive states of the metastable nonradiative recombination centers causing blinking is perturbed by strain. Reversible stochastic transformation of several nonradiative centers per microcrystal under application/release of the local stress can lead to the observed PL quenching and recovery. Fitting the experimental PL trajectories by a phenomenological model based on viscoelasticity provides a characteristic time of strain relaxation in MAPbI3 on the order of 10-100 s. The key role of metastable defect states in nonradiative losses and in the self-healing properties of perovskites is suggested.

A novel trimeric perylenediimide (PDI) macrocycle was synthesized, and its intramolecular electronic couplings were investigated by bulk and single-molecule optical spectroscopy and by various theoretical approaches. In polarization-resolved excitation spectroscopy at 1.2 K in a PMMA matrix, the appearance and disappearance of the three zero-phonon lines (ZPLs) of an individual trimer by changing the polarization in steps of 60° nicely reflect an approximate triangular geometry of the macrocycle and indicate localized excitations that are transferred by incoherent hopping processes at time scales of around 1 ps as inferred from the ZPL linewidths. The electronic coupling strength deduced from the low temperature data is found to be in good agreement with theoretical estimates. Bulk spectroscopy in toluene at room temperature indicates that the excitations are also localized under these conditions. Theory reveals that the reasons for the localized nature of the excitations at room and low temperatures are different. For a rigid macrocycle, the excitations are predicted to be delocalized, but molecular dynamics simulations point to considerable structural flexibility at ambient temperatures, which counteracts excitation delocalization. At 1.2 K in a PMMA matrix, this effect is too small to lead to localization. Yet, supported by simple model calculations, the disorder in the PMMA host induces sufficient differences between the PDI chromophores, which again result in localized excitations. By addressing crucial aspects of excitation energy transfer, our combined approach provides a detailed and quantitative account of the interchromophore communication in a trimeric macrocycle.

A series of near-infrared (NIR) organic absorbers, named FNs and FPs, have been obtained with absorption maxima from 870 nm to 1100 nm and thus falling into the attractive second near-infrared region (NIR-II). The synthesis of their extended aromatic cores utilized an initial aryl-amination between 4-aminonaphthalene-1,8-dicarboximide (NMI-NH2 ) or 9-aminoperylene-3,4-dicarboximide (PMI-NH2 ) with chloro-substituted 9,10-anthraquinones followed by a novel base-induced cyclodehydrogenation. A NIR-II pigment, compound FPP, was obtained through de-alkylation of a soluble precursor. The synthesis of this photostable pigment is high-yielding and avoids column chromatographic purification which is important for many applications.

Internal conversion (IC) often is the dominating relaxation pathway in NIR emitters, lowering their fluorescence quantum yield. Here, we investigate dibenzoterrylene (DBT) by bulk and single molecule spectroscopy. With increasing solvent polarity, the S1-S0 energy gap decreases leading to a decrease of the fluorescence quantum yield and an increase of the IC rate in full accordance with the energy gap law. Making use of the unexpectedly strong fluorescence solvatochromism of this aromatic hydrocarbon, the validity of the energy gap law could also be demonstrated at the single molecule level. The S1-S0 energy gap not only controls the fluorescence lifetime and quantum yield of single molecules but also dictates how these quantities develop during spectral fluctuations. Our results open new avenues into unexplored single molecule photophysics and appear as a promising tool for nanoscale probing of dynamic heterogeneities.

The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET (fluorescence resonance energy transfer) couple. Fluorescence correlation spectroscopy and photon-burst detection were employed to examine FRET pair labeled DENV-PRs freely diffusing in solution. The measurements were performed with two double mutants and with two dye couples. The data provide strong evidence that an equilibrium of at least two conformations of DENV-PR exists in solution. The competitive inhibitor stabilizes the closed state. Because the open and closed conformations appear to coexist in solution, our results support the picture of a conformational selection rather than that of an induced fit mechanism with respect to the inhibitor-induced formation of the closed state.

Excitation energy and charge transfer processes in perylene diimide dye-CdSe quantum dot complexes have been studied by femtosecond transient absorption spectroscopy. After excitation of the quantum dots high above the band edge, the electronically excited perylene diimide forms on a timescale similar to the ultrafast quantum dot intraband relaxation. An extraordinarily fast energy transfer time of <300 fs is determined, indicating that the transfer from the hot excitonic state competes with quantum dot intraband relaxation. A much slower kinetic component is attributed to the energy transfer from the quantum dot's lowest excitonic state to perylene diimide. At high dye-quantum dot ratios, the electronically excited dye generated in the energy transfer reaction is reduced by an electron transfer from the quantum dot ground state. This study demonstrates the potential of the investigated organic-inorganic hybrid for the design of tailor-made energy transfer systems, which can be applied in the emerging field of hot exciton utilization.

Sensitization of organic molecules by semiconductor nanocrystals is a promising way to boost the absorption of the former, important for applications in fluorescence labeling and photocatalysis. Semiconductor nanoplatelets provide the opportunity of increasing the absorption cross section by increasing the lateral dimension without inducing changes to the band gap. However, the lateral size dependence of FRET in nanoplatelet small-molecule conjugates is unknown. Our FRET study supports the view that excitons in nanoplatelets are localized in a small area and spatially distributed in a stochastic fashion. In larger nanoplatelets, the likelihood of an offset between the position of the exciton and the acceptor increases. Although this could be mitigated by increasing the number of fluorophores, excessive coverage of the nanoplatelets with the fluorophores leads to severe self-quenching. Based on these results, we predict that using few fluorophores on nanoplatelets with edge lengths of similar to 9 nm would optimize the delicate balance between maximizing the absorption cross section of the nanoplatelets and minimizing the likelihood of exciton-fluorophore offset and self-quenching simultaneously, thus leading to optimal FRET conditions.

We report the observation of ubiquitous contamination of dielectric substrates and poly(methyl methacrylate) matrices by organic molecules with optical transitions in the visible spectral range. Contamination sites of individual solvent-related fluorophores in thin films of poly(methyl methacrylate) constitute fluorescence hotspots with quantum emission statistics and quantum yields approaching 30% at cryogenic temperatures. Our findings not only resolve prevalent puzzles in the assignment of spectral features to various nanoemitters on bare dielectric substrates or in polymer matrices but also identify the means for the simple and cost-efficient realization of single-photon sources in the visible spectral range.

2-Iminopyrroles [HtBuL, 4-tert-butyl phenyl(pyrrol-2ylmethylene) amine] are non-fluorescent p systems. However, they display blue fluorescence after deprotonation with alkali metal bases in the solid state and in solution at room temperature. In the solid state, the alkali metal 2-imino pyrrolates, M(L-tBu), aggregate to dimers, [M(L-tBu)(NCR)](2) (M= Li, R= CH3, CH(CH3)CNH2), or polymers, [M(L-tBu)](n) (M= Na, K). In solution (solv= CH3CN, DMSO, THF, and toluene), solvated, uncharged monomeric species M(L-tBu)(solv)(m) with N, N'-che-lated alkali metal ions are present. Due to the electron-rich pyrrolate and the electron-poor arylimino moiety, the M(L-tBu) chromophore possesses a low-energy intraligand chargetransfer (ILCT) excited state. The chelated alkali cations rigidify the chromophore, restricting intramolecular motions (RIM) by the chelation-enhanced fluorescence (CHEF) effect in solution and, consequently, switch-on a blue fluorescence emission.