Membraneless organelles (MLOs) are cellular biomolecular condensates formed by liquid-liquid phase separation. Their biological functions are intimately linked to their material properties including viscosity. Condensate viscosity is determined by the size, shape, concentration and molecular interactions between MLOs components. It impacts the diffusion of MLOs constituents and the selective permeability of the condensate, thereby regulating the rate of biochemical reactions. Viscosity modifications associated with liquid-to-gel transition of the condensates are related to pathologies. Current experimental approaches for characterizing the material properties of cellular condensates remain limited. In this study, we report the use of BODIPY-based molecular rotors, in combination with fluorescence lifetime imaging microscopy (FLIM), to monitor the microviscosity of cellular MLOs directly in living cells. The fluorescence lifetime of BODIPY derivatives increases with the viscosity of their microenvironment, enabling quantitative assessment of microviscosity within condensates. HaloTag technology was employed to specifically label MLO components. Our findings reveal that the nucleolus exhibits higher viscosity than the surrounding nucleoplasm and that microviscosity varies across nucleolar sub-compartments. Furthermore, nucleolar reorganization induced by inhibition of rRNA synthesis results in a measurable increase in microviscosity. Finally, we demonstrate that the microviscosity of stress granules is lower than that of the nucleolus. Overall, presented results demonstrate the strong potential of the BODIPY based molecular rotors as a versatile and powerful tools for probing the material properties of cellular MLOs. ### Competing Interest Statement The authors have declared no competing interest. French National Research Agency (ANR), ANR-23-CE11-0016 FluoLLPS, ANR-24-INBS-0005 FBI BIOGEN Unistra IdEx, R22086MM

BODIPY dyes are widely used in bioimaging, yet their aggregation behavior within the plasma membrane (PM) remains poorly exploited for single-molecule localization microscopy (SMLM). Here, we design a series of green-emitting PM-targeted BODIPY dimers engineered to undergo spontaneous and transient H- and J-aggregation. By tuning the linker length between the fluorophores, we identify dimers that form intramolecular H-aggregates in polar media and emissive J-aggregates (λem ≈ 535 nm) through membrane-driven intermolecular interactions. In lipid bilayers, all dimers aggregate above a probe/lipid ratio of 1/100, exclusively generating J-aggregates. In live-cell SMLM, the monomeric MB-488 provides high event numbers via diffusion-driven emission, whereas dimers exhibit stable blinking and yield brighter red-shifted J-aggregate events with improved localization precision. Red-channel events localize to specific PM regions, suggesting preferential J-aggregation within distinct membrane microdomains. HaloTag constructs targeted to cell-surface PDGFR further confirm that intramolecular J-aggregation is possible but strongly amplified by the membrane through intermolecular collisions. These results demonstrate that green BODIPYs and their J-aggregates enable robust live SMLM in green and red channels, and reveal the PM as a privileged environment for emissive J-aggregation. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, 5D-SURE ANR-21-CE42-0015

The human immunodeficiency virus 1 (HIV-1) group specific antigen (Gag) polyprotein, the main structural protein of HIV-1 is sufficient to mimic the late stages of the viral replication cycle. When expressed in cells, Gag binds to RNAs and assembles at the plasma membrane to form virus-like particles (VLPs) with a morphology similar to that of HIV-1 virions. The nucleocapsid (NC) domain of Gag plays a critical role in RNA binding and selective encapsidation of the viral genome. This work focuses on the impact of NC deletion on Gag assembly, VLP formation, and intracellular trafficking. By combining several fluorescence-based quantitative microscopy techniques, we show that in the absence of the NC domain, Gag cytoplasmic oligomerization and formation of initial ribonucleoprotein complexes are impacted, resulting in a significant delay in the kinetics of VLP formation. Interestingly, VLPs formed from the Gag-ΔNC mutant display greater diversity in size and shape. Single particle tracking experiments revealed that VLPs formed at the plasma membrane are immobile, while intracellular VLPs are mostly mobile. For the latter, the motions and diffusion coefficients of VLPs formed by the Gag-ΔNC mutant were highly similar to those of VLPs formed with the wild-type Gag, indicating that the NC domain is not implicated in the intracellular trafficking. These findings illustrate how fluorescence-based microscopy techniques can provide quantitative insights into the role of the NC domain in Gag assembly.

ABSTRACT Molecular aggregation profoundly alters the optical properties of fluorophores, yet its behavior in dynamic biological environments remains insufficiently understood and rarely harnessed functionally. Here, we report a membrane‐controlled aggregation strategy based on plasma membrane‐targeted BODIPY dimers engineered to undergo tunable H‐ and J‐aggregation. By modulating the linker length, we establish a clear structure–aggregation relationship in which dimers form intramolecular H‐aggregates in polar media, while two‐dimensional confinement within lipid bilayers promotes intermolecular aggregation with red‐shifted emission. Quantitative studies in model membranes reveal a threshold‐dependent transition toward aggregate formation above a probe/lipid ratio of 1/100, highlighting the lipid bilayer as an active supramolecular platform that enhances probe‐probe encounters and favors emissive excitonic states. In live cells, this membrane‐promoted J‐aggregation generates spontaneous blinking behavior that enables dual‐channel single‐molecule localization microscopy without specialized imaging buffers. Mechanistic investigations using HaloTag constructs confirm that while intramolecular aggregation can occur, membrane‐driven intermolecular collisions strongly amplify J‐aggregate formation. These findings demonstrate that biological membranes can serve as dynamic two‐dimensional reactors for excitonic coupling, and establish membrane‐induced J‐aggregation of small‐molecule fluorophores as a functional and generalizable principle for bioimaging.

This study investigates the blinking behavior of dye-loaded organic nanoparticles (NPs) and elucidates the role of energy transfer (EET) and dye aggregation. Using spectrally resolved single-molecule microscopy, we characterize the optical properties of NPs composed of poly(lactic-co-glycolic acid) (PLGA) and poly(methyl methacrylate) (PMMA) with varying dye loadings. At high dye concentrations, the NPs exhibit intensity fluctuations that are attributed to transient species quenching the emission. Temperature dependent studies evidence that the quenching disappears at 77 K, highlighting the role of dye diffusion in the polymer matrix. Spectral analysis confirms the formation of red-shifted emissive aggregates and nonemissive aggregates as major contributors to the blinking mechanism. Computational studies support these findings, identifying stable crossed J-dimers responsible for red-shifted emission and nonemissive H-dimers associated with fluorescence quenching. This work improves our understanding of the collective behavior of dyes embedded in polymeric NPs, paving the way for the development of brighter and more efficient fluorescent nanomaterials.

Fascinating process of light harvesting (LH) in plants and bacteria leads to photosynthesis of oxygen and organic matter on the planet. It inspires researchers in the last decades to develop synthetic analogues - artificial LH nanomaterials. Here, LH nanomaterials based on dyes (organic chromophores) are reviewed. The fundamental aspects of LH include dye assembly into materials with minimal energy losses (i.e., high fluorescence quantum yield with minimized aggregation-caused quenching) and fast excitation energy transfer with large exciton migration length. The efficient energy transfer from LH nanomaterial to an acceptor leads to an amplification of acceptor emission - antenna effect, which constitutes the key performance parameter of LH nanoantenna. Individual classes of dye-based LH nanomaterials are analyzed: covalent molecular arrays of dyes (e.g., dendrimers and macrocycles), aggregates of "classical" dyes, aggregation-induced emission nanomaterials, metal-organic frameworks, ion-associated nanomaterials, and hybrid dye-based organic systems, such as dye-biomolecule hybrids, micelles and supramolecular polymers, dye-loaded silica and polymeric nanoparticles. The design principles and key performance characteristics of their selected (non-exhaustive) examples are analyzed. Due to their capacity to amplify the acceptor fluorescence signal, LH nanomaterials are particularly suitable for amplified sensing of small molecules, ions, biomolecules, which is particularly attractive for biomedical applications.

Exciton dynamics play a crucial role in determining the efficiency of organic photovoltaic devices and photo-detectors. However, establishing clear correlations between molecular structure and exciton diffusion length remains a significant challenge, limiting the rational design of more efficient materials. In this study, we investigate exciton transport in thin films of a planar dumbbell-shaped electron donor composed of discotic triazatruxene end-groups and an electron-deficient central unit. These molecules self-assemble into unique bridged-columnar structures, which are known to support efficient charge transport, although their impact on exciton dynamics had not yet been explored. Using a combination of time-resolved photoluminescence (TRPL), spatially resolved TRPL, and exciton-exciton annihilation measurements, we examine how structural order influences exciton diffusion in both the columnar-nematic and crystalline phases. We show that crystallization leads to a twofold increase in exciton diffusion length, reaching values comparable to those observed in state-of-the-art non-fullerene acceptors. Although the molecules exhibit a typical Stokes shift that is not particularly favorable for F & ouml;rster energy transfer (FRET), efficient exciton transport is nonetheless achieved-enabled by long exciton lifetimes and anisotropic energy transfer within its distinctive bridged-columnar architecture. These results, supported by FRET analysis, highlight the effectiveness of the molecule's tailored dumbbell-shaped design and its ability to self-assemble into ordered structures that support both long-range exciton diffusion and efficient charge mobility.

Characterization of nanoscale formulations is a continuous challenge. Size, morphology and surface properties are the most common characterizations. However, physicochemical properties inside the nanoparticles, like viscosity, cannot be directly measured. Herein, we propose an original approach to measuring dynamic viscosity using a lipidic molecular rotor solubilized in the core of nano-formulations. These molecules undergo conformational changes in response to viscosity variations, leading to observable changes in fluorescence intensity and lifetime, able to sense the volume properties of dispersed nano-domains. The lipophilic molecular rotor (BOPIDY derivatives) was specifically synthesized and characterized as oil viscosity sensing in large volumes. A second part of the study compares these results with rBDP-Toco in nano-emulsions. The objective is to evaluate the impact of the formulation, droplet size and composition on the viscosity of the droplet's core. The lipophilic rotor showed a universal behavior whatever the oil composition, giving a master curve. Applied to nano-formulations, it reveals the viscosity inside the nano-emulsion droplets, enabling the detection of slight variations between reference oil samples and the nano-formulated ones. This new tool opens the way to the fine characterization of complex colloids and multi-domain nano and micro systems, potentially applied to hybrid materials and biomaterials.

To fully exploit the potential of isothiazologuanosine (tzG), an isomorphic and isofunctional fluorescent analogue of guanosine, as a probe for DNA and RNA, we characterized its photophysics and in particular its excited-state reactions over a wide pH range (-0.6 to 12) and time scale (100 fs-100 ns) by combining transient absorption and time-correlated single photon counting measurements with quantum mechanical calculations. At acidic pH, the dominant ground-state species tzG-H1-H3+, where the N atoms in positions 1 and 3 are protonated, rapidly converts to the more stable tautomer tzG-H1-H7+ in its excited state. The latter then deprotonates to form the tzG-H1 neutral species with an excited-state pKa* value that differs by three pH units from the ground-state pKa value. The rate constants governing the excited-state reactions and the fluorescence lifetime of each species were all determined. With the exception of intramolecular and solvent relaxations, no excited-state reactions in the femtosecond to nanosecond time scale were, however, observed between the dominant tzG-H1 and tzG-H3 tautomers in equilibrium at neutral pH or for tzG-H1 deprotonation at high pH. Because of the distinct spectra, fluorescence quantum yields and lifetimes of its different protonated and deprotonated forms, tzG is highly responsive over a wide range of acidic (0-5) and basic pH values (8-10). The mechanisms revealed herein will be instrumental for tzG-labelled oligonucleotides in order to interpret proton transfer reactions as well as interactions with specific protein domains, which, due to local electrostatic changes and water exclusion effects, may shift the pKa values of tzG to a more physiologically relevant range.

Light-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 approximate to 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. A previously introduced light-harvesting nanoantenna, based on polymeric nanoparticles loaded with rhodamine dyes and bulky hydrophobic counterions, provides an antenna effect of approximate to 1000. Here, blank hydrophobic salts (BHS) formed by the same bulky counterion with an optically inactive cation are found to suppress aggregation-cased quenching (ACQ) and fluorescence blinking within nanoparticles, leading to the record-breaking antenna effect of 4800. image

The viscosity that ensures the controlled diffusion of biomolecules in cells is a crucial biophysical parameter. Consequently, fluorescent probes capable of reporting viscosity variations are valuable tools in bioimaging. In this field, red-shifted probes are essential, as the widely used and gold standard probe remains green-emitting molecular rotors based on BODIPY. Here, we demonstrate that pyrrolyl squaraines, red-emissive fluorophores, exhibit high sensitivity over a wide viscosity range from 30 to 4890 mPa·s. Upon alkylation of the pyrrole moieties, the probes improve their sensitivity to viscosity through an enhanced twisted intramolecular charge transfer phenomenon. We utilized this scaffold to develop a plasma membrane probe, pSQ-PM, that efficiently stains the plasma membrane in a fluorogenic manner. Using fluorescence lifetime imaging, pSQ-PM enabled efficient sensing of viscosity variations in the plasma membrane under various conditions and in different cell lines (HeLa, U2OS, and NIH/3T3). Moreover, upon incubation, pSQ-PM stained the membrane of intracellular vesicles and suggested that the lysosomal membranes displayed enhanced fluidity.

DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti–Pol II antibody–conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.

HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.

Photoactivatable fluorescent probes are valuable tools in bioimaging for tracking cells down to single molecules and for single molecule localization microscopy. For the latter application, green emitting dyes are in demand. We herein developed an efficient green-emitting photoactivatable furanyl-BODIPY (PFB) and we established a new mechanism of photoactivation called Directed Photooxidation Induced Activation (DPIA) where the furan is photo-oxidized in a directed manner by the singlet oxygen produced by the probe. The efficient photoconverter (93-fold fluorescence enhancement at 510 nm, 49 % yield conversion) is functionalizable and allowed targeting of several subcellular structures and organelles, which were photoactivated in live cells. Finally, we demonstrated the potential of PFB in super-resolution imaging by performing PhotoActivated Localization Microscopy (PALM) in live cells.

Efficient exciton transport is the essential property of natural and synthetic light-harvesting (LH) devices. Here we investigate exciton transport properties in LH organic polymer nanoparticles (ONPs) of 40 nm diameter. The ONPs are loaded with a rhodamine B dye derivative and bulky counterion, enabling dye loadings as high as 0.3 M, while preserving fluorescence quantum yields larger than 30%. We use time-resolved fluorescence spectroscopy to monitor exciton-exciton annihilation (EEA) kinetics within the ONPs dispersed in water. We demonstrate that unlike the common practice for photoluminescence investigations of EEA, the non-uniform intensity profile of the excitation light pulse must be taken into account to analyse reliably intensity-dependent population dynamics. Alternatively, a simple confocal detection scheme is demonstrated, which enables (i) retrieving the correct value for the bimolecular EEA rate which would otherwise be underestimated by a typical factor of three, and (ii) revealing minor EEA by-products otherwise unnoticed. Considering the ONPs as homogeneous rigid solutions of weakly interacting dyes, we postulate an incoherent exciton hoping mechanism to infer a diffusion constant exceeding 0.003 cm2 s-1 and a diffusion length as large as 70 nm. This work demonstrates the success of the present ONP design strategy at engineering efficient exciton transport in disordered multichromophoric systems. The unbiased fluorescence monitoring of exciton-exciton annihilation kinetics reveals an exciton diffusion length exceeding 70 nm in highly concentrated, disordered, dye-loaded organic nanoparticles.

The realm of self-healing materials integrates chemical and physical mechanisms that prevent wear and fracturing and extend the operational lifetime. Unlike the favorable rheology of amorphous soft materials that facilitates efficient contact between fragments, the efficiency of recovery of atomistically ordered materials is restricted by slower interfacial mass transport and the need for ideal physical alignment, which limits their real-world application. We report drastic enhancements in efficiency and recovery time in the self-healing of anilinium bromide, challenging these limitations. Crystals of this material recovered up to 49% within seconds and up to 95% after 100 min via ferroelastic detwinning. The spatial evolution of strain during cracking and healing was measured in real time using digital image correlation. Favorable alignment and strong ionic bonding across the interface of partially fractured crystals facilitate self-healing. This study elevates organic crystals close to the best-in-class self-healing polymers and sets an approach for durable crystal-based optoelectronics. Self-healing in structurally ordered materials is restricted by slow interfacial mass transport and the need for ideal physical alignment. Here the authors show self-healing in an anilinium bromide crystals achieving up to 95% recovery through ferroelastic detwinning.

Application of fluorescence techniques to investigate molecular interactions with nucleic acids is complicated by their poor emission, making the substitution of natural nucleobases by fluorescent nucleoside analogues (FNAs) a useful strategy. A breakthrough in fluorescent nucleoside analogues has been the development of thienoguanosine (thG) and isothiazologuanosine (tzG), two isosteric mimics of guanosine (G). Due to its N7 atom needed in Hoogsteen base pairs and enzyme recognition, tzG is also an isofunctional G surrogate. Herein, we integrated fluorescence spectroscopy measurements with quantum mechanical (QM) calculations to characterize the mechanisms underlying tzG photophysics in different solvents. In dioxane and ethyl acetate, tzG existed primarily as a H1 keto-amino tautomer with short fluorescence lifetime (r - 2 ns) and low quantum yield (O - 0.10). In buffer, the H1 tautomer (O = 0.36, r = 8.84 ns) coexisted with a weakly emissive H3 keto-amino tautomer. The two tautomers were also observed in methanol, but with a 30% decrease in O and r values for the major H1 tautomer. QM calculations suggested that the main non-radiative pathway of tzG-H1 involves NS bond loosening and is responsible for the more solvent-sensitive O and r values compared to thG. This pathway is much more efficient for tzG-H3, for which an additional pathway to a dark n pi* state and a large coupling with triplet states further explain its very low emission. This study lays the ground for rationally using tzG as a sensitive FNA.

Photomodulable fluorescent probes are drawing increasing attention due to their applications in advanced bioimaging. Whereas photoconvertible probes can be advantageously used in tracking, photoswitchable probes constitute key tools for single-molecule localization microscopy to perform super-resolution imaging. Herein, we shed light on a red and far-red BODIPY, namely, BDP-576 and BDP-650, which possess both properties of conversion and switching. Our study demonstrates that these pyrrolyl-BODIPYs convert into typical green- and red-emitting BODIPYs that are perfectly adapted to microscopy. We also showed that this pyrrolyl-BODIPYs undergo Directed Photooxidation Induced Conversion, a photoconversion mechanism that we recently introduced, where the pyrrole moiety plays a central role. These unique features were used to develop targeted photoconvertible probes toward different organelles or subcellular units (plasma membrane, mitochondria, nucleus, actin, Golgi apparatus, etc.) using chemical targeting moieties and a Halo tag. We notably showed that BDP-650 could be used to track intracellular vesicles over more than 20 min in two-color imagings with laser scanning confocal microscopy, demonstrating its robustness. The switching properties of these photoconverters were studied at the single-molecule level and were then successfully used in live single-molecule localization microscopy in epithelial cells and neurons. Both membrane- and mitochondria- targeted probes could be used to decipher membrane 3D architecture and mitochondrial dynamics at the nanoscale. This study builds a bridge between the photoconversion and photoswitching properties of probes undergoing directed photooxidation and shows the versatility and efficacy of this mechanism in advanced live imaging.

Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly.

Invited for the cover of this issue is the group of Mayeul Collot at the University of Strasbourg (CNRS). The image depicts the effect of simple chemical tuning on coumarin dyes to tune and improve the DPIC photoconversion mechanism. Read the full text of the article at 10.1002/chem.202203933.