Stable Super-Resolution Imaging of Cell Membrane Nanoscale Subcompartment Dynamics with a Buffering Cyanine Dye.

Super-resolution fluorescence imaging is a crucial method for visualizing the dynamics of the cell membrane involved in various physiological and pathological processes. This requires bright fluorescent dyes with excellent photostability and labeling stability to enable long-term imaging. In this context, we introduce a buffering-strategy-based cyanine dye, SA-Cy5, designed to identify and label carbonic anhydrase IX (CA IX) located in the cell membrane. The unique feature of SA-Cy5 lies in its ability to overcome photobleaching. When the dye on the cell membrane undergoes photobleaching, it is rapidly replaced by an intact probe from the buffer pool outside the cell membrane. This dynamic replacement ensures that the fluorescence intensity on the cell membrane remains stable over time. Under the super-resolution structured illumination microscopy (SIM), the cell membrane can be continuously imaged for 60 min with a time resolution of 20 s. This extended imaging period allows for the observation of substructural dynamics of the cell membrane, including the growth and fusion of filamentous pseudopodia and the fusion of vesicles. Additionally, this buffering strategy introduces a novel approach to address the issue of poor photostability associated with the cyanine dyes.

Solvatochromic Buffering Fluorescent Probe Resolves the Lipid Transport and Morphological Changes during Lipid Droplet Fusion by Super-Resolution Imaging.

The varied functions of lipid droplets, which encompass the regulation of lipid and energy homeostasis, as well as their association with the occurrence of various metabolic diseases, are intricately linked to their dynamic properties. Super-resolution imaging techniques have emerged to decipher physiological processes and molecular mechanisms on the nanoscale. However, achieving long-term dynamic super-resolution imaging faces challenges due to the need for fluorescent probes with high photostability. This paper introduces LD-CF, a "buffering probe" for imaging lipid droplet dynamics using structured illumination microscopy (SIM). The polarity-sensitive LD-CF eliminates background fluorescence with a "cyan filter" strategy, enabling wash-free imaging of lipid droplets. In the fluorescent "off" state outside droplets, the probes act as a "buffering pool", replacing photobleached probes inside droplets and enabling photostable long-term SIM imaging. With this probe, three modes of lipid droplet fusion were observed, including the discovery of fusion from large to small lipid droplets. Fluorescence intensity tracking also revealed the direction of lipid transport during the lipid droplet fusion.

Super-resolution imaging reveals the mechanism of endosomal acidification inhibitors against SARS-CoV-2 infection.

In this study, super-resolution structured illumination microscope (SIM) was used to analyze molecular mechanism of endocytic acidification inhibitors in the SARS-CoV-2 pandemic, such as Chloroquine (CQ), Hydroxychloroquine (HCQ) and Bafilomycin A1 (BafA1). We fluorescently labeled the SARS-CoV-2 RBD and its receptor ACE2 protein with small molecule dyes. Utilizing SIM imaging, the real-time impact of inhibitors (BafA1, CQ, HCQ, Dynasore) on the RBD-ACE2 endocytotic process was dynamically tracked in living cells. Initially, the protein activity of RBD and ACE2 was ensured after being labeled. And then our findings revealed that these inhibitors could inhibit the internalization and degradation of RBD-ACE2 to varying degrees. Among them, 100 nM BafA1 exhibited the most satisfactory endocytotic inhibition (~63.9%) and protein degradation inhibition (~97.7%). And it could inhibit the fusion between endocytic vesicles in the living cells. Additionally, Dynasore, a widely recognized dynein inhibitor, also demonstrated cell acidification inhibition effects. Together, these inhibitors collectively hinder SARS-CoV-2 infection by inhibiting both the viral internalization and RNA release. The comprehensive evaluation of pharmacological mechanisms through super-resolution fluorescence imaging has laid a crucial theoretical foundation for the development of potential drugs to treat COVID-19.

Spontaneously Blinking Rhodamine Dyes for Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) has found extensive applications in various fields of biology and chemistry. As a vital component of SMLM, fluorophores play an essential role in obtaining super-resolution fluorescence images. Recent research on spontaneously blinking fluorophores has greatly simplified the experimental setups and extended the imaging duration of SMLM. To support this crucial development, this review provides a comprehensive overview of the development of spontaneously blinking rhodamines from 2014 to 2023, as well as the key mechanistic aspects of intramolecular spirocyclization reactions. We hope that by offering insightful design guidelines, this review will contribute to accelerating the advancement of super-resolution imaging technologies.

Directional Construction of Active Naphthalenic Species within SAPO-34 Crystals toward More Efficient Methanol-to-Olefin Conversion.

Olefin selectivity and catalyst lifetime are two key metrics for industrial methanol-to-olefin catalysts. Currently, it is very difficult to obtain high olefin selectivity and long catalyst lifetime at the same time. Herein, a feasible strategy combining precoking and steaming to directionally construct the active naphthalenic species within the crystal center of the SAPO-34 catalyst has been developed, which can not only promote the lower olefin selectivity to ∼89% (ethylene and propylene) but also prolong the catalyst lifetime by ∼3.7-fold in the methanol-to-olefin conversion. Structured illumination microscopy, in situ ultraviolet-visible spectroscopy, and online mass spectrometry elucidate the spatiotemporal distribution and evolution of the naphthalenic species during the precoking and steaming processes. This one-stone-two-birds strategy is applicable to a commercial SAPO-34 catalyst containing a binder, demonstrating its bright prospect in the methanol-to-olefin industry.

Molecular origins of the multi-donor strategy in inducing bathochromic shifts and enlarging Stokes shifts of fluorescent proteins.

Long-wavelength fluorescent proteins (LWFPs) and LWFP-based sensors are indispensable tools for bioimaging and biosensing applications. However, it remains challenging to develop LWFPs with outstanding brightness and/or sensitivities, largely due to the lack of simple and effective molecular design strategies. Herein, we rationalized the molecular origins of a multi-donor strategy that affords significant bathochromic shifts and large Stokes shifts with minimal structural changes in the resulting protein fluorophores. We analyzed three key factors that affect the spectral properties of these fluorophores, including the (1) substituent position, (2) electron-donating strength, and (3) number of electron-donating groups. We further demonstrated that this simple design strategy is generalizable to various fluorophore families. We expect that this work can provide rational guidelines for developing fluorescent proteins (and small-molecule fluorophores) with long emission wavelengths and large Stokes shifts.

Twisted intramolecular charge transfer (TICT) and twists beyond TICT: from mechanisms to rational designs of bright and sensitive fluorophores.

The twisted intramolecular charge transfer (TICT) mechanism has guided the development of numerous bright and sensitive fluorophores. This review briefly overviews the history of establishing the TICT mechanism, and systematically summarizes the molecular design strategies in modulating the TICT tendency of various organic fluorophores towards different applications, along with key milestone studies and representative examples. Additionally, we also succinctly review the twisted intramolecular charge shuttle (TICS) and twists during photoinduced electron transfer (PET), and compare their similarities and differences with TICT, with emphasis on understanding the structure-property relationships between the twisted geometries and how they can directly affect the fluorescence of the molecules. Such structure-property relationships presented herein will greatly aid the rational development of fluorophores that involve molecular twisting in the excited state.

Revisiting imidazolium receptors for the recognition of anions: highlighted research during 2010-2019.

Imidazolium based receptors selectively recognize anions, and have received more and more attention. In 2006 and 2010, we reviewed the mechanism and progress of imidazolium salt recognition of anions, respectively. In the past ten years, new developments have emerged in this area, including some new imidazolium motifs and the identification of a wider variety of biological anions. In this review, we discuss the progress of imidazolium receptors for the recognition of anions in the period of 2010-2019 and highlight the trends in this area. We first classify receptors based on motifs, including some newly emerging receptors, as well as new advances in existing receptor types at this stage. Then we discuss separately according to the types of anions, including ATP, GTP, DNA and RNA.

Exploring the Influence of Inter- and Intra-crystal Diversity of Surface Barriers in Zeolites on Mass Transport by Using Super-Resolution Microimaging of Time-Resolved Guest Profiles.

The applications of nanoporous crystalline materials are closely related to the mass transfer of guest molecules. However, the fundamental knowledge of mass transfer, and in particular the surface barriers controlled by the permeation of guest molecules through the external surfaces of materials, is still incomplete. The diversity of surface permeability at the single-crystal level, caused by the varying origins of surface transport resistance, hinders the rational materials design and needs better understanding. Herein, we probe the molecular transport in single zeolite crystals with fluorescent 4-(4-diethylaminostyryl-1-methylpyridinium iodide) (DAMPI) using super-resolution structured illumination microscopy (SIM). It showed that both the inter- and intra-crystal diversity of surface barriers could be monitored by detecting the diffusion behaviors on the center and surface planes in single crystals. This adds a new perspective for studying the origins of the surface barriers as well as the molecular transport mechanisms in nanoporous materials.

In Situ Real-Time Nanoscale Resolution of Structural Evolution and Dynamics of Fluorescent Self-Assemblies by Super-Resolution Imaging.

The visualization of self-assembled structure and dynamics at the molecular level has become a powerful method to understand structure-function relationships of self-assembly. Herein, we in situ real-time imaged the dynamic process of benzyl-naphthalimide dyes at the nanoscale and inspected their internal structure with minimum 2.8 nm localization accuracy through single-molecule localization microscopy (SMLM) imaging. We monitored the growth process of three different assemblies in situ, which possessed highly heterogeneous dynamics with different shapes and growth rates. Furthermore, diverse growth rates were also found at different sites in the same assembly. These results highlight the application of super-resolution microscopy techniques for real-time visualization of internal assembled structure and dynamics in situ.

An Acid-Regulated Self-Blinking Fluorescent Probe for Resolving Whole-Cell Lysosomes with Long-Term Nanoscopy.

Long-term super-resolution imaging appears to be increasingly important for unraveling organelle dynamics at the nanoscale, but is challenging due to the need for highly photostable and environment-sensitive fluorescent probes. Here, we report a self-blinking fluorophore that achieved 12 nm spatial resolution and 20 ms time resolution under acidic lysosomal conditions. This fluorophore was successfully applied in super-resolution imaging of lysosomal dynamics over 40 min. The pH dependence of the dye during blinking made the fluorophore sensitive to lysosomal pH. This probe enables simultaneous dynamic and pH recognition of all lysosomes in the entire cell at the single-lysosome-resolved level, which allowed us to resolve whole-cell lysosome subpopulations based on lysosomal distribution, size, and luminal pH. We also observed a variety of lysosome movement trajectories and different types of interactions modes between lysosomes.

A Descriptor for Accurate Predictions of Host Molecules Enabling Ultralong Room-Temperature Phosphorescence in Guest Emitters.

Although doping can induce room-temperature phosphorescence (RTP) in heavy-atom free organic systems, it is often challenging to match the host and guest components to achieve efficient intersystem crossing for activating RTP. In this work, we developed a simple descriptor ΔE to predict host molecules for matching the guest RTP emitters, based on the intersystem crossing via higher excited states (ISCHES) mechanism. This descriptor successfully predicted five commercially available host components to pair with naphthalimide (NA) and naphtho[2,3-c]furan-1,3-dione (2,3-NA) emitters with a high accuracy of 83 %. The yielded pairs exhibited bright yellow and green RTP with the quantum efficiency up to 0.4 and lifetime up to 1.67 s, respectively. Using these RTP pairs, we successfully achieved multi-layer message encryption. The ΔE descriptor could provide an efficient way for developing doping-induced RTP materials.

Rapid Enzyme-Mediated Biotinylation for Cell Surface Proteome Profiling.

Cell surface is the primary site for sensing extracellular stimuli. The knowledge of the transient changes on the surfaceome upon a perturbation is very important as the initial changed proteins could be driving molecules for some phenotype. In this study, we report a fast cell surface labeling strategy based on peroxidase-mediated oxidative tyrosine coupling strategy, enabling efficient and selective cell surface labeling within seconds. With a labeling time of 1 min, 2684 proteins, including 1370 (51%) cell surface-annotated proteins (cell surface/plasma membrane/extracellular), 732 transmembrane proteins, and 81 cluster of differentiation antigens, were identified from HeLa cells. By comparison with the negative control experiment using quantitative proteomics, 500 (68%) out of the 731 significantly enriched proteins (p-value < 0.05, ≥2-fold) in positive experimental samples were cell surface-annotated proteins. Finally, this technology was applied to track the dynamic changes of the surfaceome upon insulin stimulation at two time points (5 min and 2 h) in HepG2 cells. Thirty-two proteins, including INSR, CTNNB1, TFRC, IGF2R, and SORT1, were found to be significantly regulated (p-value < 0.01, ≥1.5-fold) after insulin exposure by different mechanisms. We envision that this technique could be a powerful tool to analyze the transient changes of the surfaceome with a good time resolution and to delineate the temporal and spatial regulation of cellular signaling.

Thermal equilibria between conformers enable highly reliable single-fluorophore ratiometric thermometers.

Fluorescent thermometers based on organic dyes play an important role in the visualization of dynamic temperature topography with high resolution. Many molecular thermometers contain two fluorophores with different temperature responses to achieve ratiometric temperature detection. However, the stability and reliability of such ratiometric thermometers are highly dependent on the susceptibility of the fluorophores towards photobleaching. Despite the use of single-fluorophore ratiometric thermometers in previous reports, the difficulty and complexity in their molecular design and synthesis severely hinder their widespread applicability. In this work, we have investigated the use of commercially available dyes (i.e., BD140 and LD688) with conformational isomers as ratiometric thermometers. Each of these dyes consists of a mixture of conformers with distinct UV-vis absorption and emission spectra. The thermal equilibrium of these conformers successfully enabled the development of ratiometric thermometers with good stability and reliability.

Stable Super-Resolution Imaging of Lipid Droplet Dynamics through a Buffer Strategy with a Hydrogen-Bond Sensitive Fluorogenic Probe.

Although super-resolution imaging offers an opportunity to visualize cellular structures and organelles at the nanoscale level, cellular heterogeneity and unpredictability still pose a significant challenge in the dynamic imaging of live cells. It is thus vital to develop better-performing and more photostable probes for long-term super-resolution imaging. Herein, we report a probe, LD-FG, for imaging lipid droplet (LD) dynamics using structured illumination microscopy (SIM). LD-FG allows wash-free imaging of LDs, owing to a hydrogen-bond sensitive fluorogenic response. The replacement of photobleached LD-FG by intact probe molecules outside the LDs ensures the long-time stability of the fluorescence imaging. With this buffering fluorogenic probe, fast and unpredictable dynamic processes of LDs can be visualized. Using this probe, two LD coalescence modes were discovered. The dynamic imaging also allowed us to propose a new model of LD maturation during adipocyte differentiation, i.e., a fast LD coalescence followed by a slow ripening step. The excellent performance of LD-FG makes the buffer strategy an effective method for designing fluorescent probes for cell dynamic imaging.

A General Descriptor ΔE Enables the Quantitative Development of Luminescent Materials Based on Photoinduced Electron Transfer.

Photoinduced electron transfer (PET) is one of the most important mechanisms for developing fluorescent probes and biosensors. Quantitative prediction of the quantum yields of these probes and sensors is crucial to accelerate the rational development of novel PET-based functional materials. Herein, we developed a general descriptor (ΔE) for predicting the quantum yield of PET probes, with a threshold value of ∼0.6 eV. When ΔE < ∼0.6 eV, the quantum yield is low (mostly <2%) due to the substantial activation of PET in polar environments; when ΔE > ∼0.6 eV, the quantum yield is high because of the inhibition of PET. This simple yet effective descriptor is applicable to a wide range of fluorophores, such as BODIPY, fluorescein, rhodamine, and Si-rhodamine. This ΔE descriptor enables us not only to establish new applications for existing PET probes but also to quantitatively design novel PET-based fluorophores for wash-free bioimaging and AIEgen development.

Descriptor ΔGC-O Enables the Quantitative Design of Spontaneously Blinking Rhodamines for Live-Cell Super-Resolution Imaging.

Herein, we reported a simple, fast, and quantitative theoretical descriptor ΔGC-O that allows accurate predictions of a wide range of spontaneously blinking rhodamines. ΔGC-O denotes the Gibbs free energy differences between the closed and open forms of rhodamines and has a good linear relationship with experimental pKcycl values. This correlation affords an effective guide for the quantitative designs of spontaneously blinking rhodamines and eliminates trial-and-error. We have validated the predictive power of ΔGC-O via the development of two spontaneously blinking rhodamines of different colors and enhanced brightness. We also demonstrated their super-resolution imaging utilities in dynamic live-cell imaging. We expect that ΔGC-O will greatly facilitate the efficient creations of spontaneously blinking fluorophores and aid the advancements of super-resolution bioimaging techniques.

Water-Induced Structural Dynamic Process in Molecular Sieves under Mild Hydrothermal Conditions: Ship-in-a-Bottle Strategy for Acidity Identification and Catalyst Modification.

Water is the most important substance in nature. Imitating the formation of natural materials, molecular sieves have been synthesized under hydrothermal conditions and applied in industry. Herein, we reveal an unforeseen observation on a very special water-induced structural dynamic process of these materials. Dynamic and reversible breaking and forming of T-O-T bonds in silicoaluminophosphate (SAPO) occurs through interactions between gaseous water and the molecular-sieve framework under mild hydrothermal conditions and is confirmed by detection of the incorporation of 17 O from H2 17 O into molecular-sieve framework. Encapsulation of the bulky molecules trimethylphosphine and pyridine (kinetic diameters much larger than the pore size of SAPO-34) into CHA cavities consolidated the water-induced dynamic process. Consequently, new insights into the dynamic features of molecular sieves in water are provided. The ship-in-a-bottle strategy based on these findings also open new fields for fine acidity identification and gives extra boost in shape-selective catalysis.

Quantitative Design of Bright Fluorophores and AIEgens by the Accurate Prediction of Twisted Intramolecular Charge Transfer (TICT).

Inhibition of TICT can significantly increase the brightness of fluorescent materials. Accurate prediction of TICT is thus critical for the quantitative design of high-performance fluorophores and AIEgens. TICT of 14 types of popular organic fluorophores were modeled with time-dependent density functional theory (TD-DFT). A reliable and generalizable computational approach for modeling TICT formations was established. To demonstrate the prediction power of our approach, we quantitatively designed a boron dipyrromethene (BODIPY)-based AIEgen which exhibits (almost) barrierless TICT rotations in monomers. Subsequent experiments validated our molecular design and showed that the aggregation of this compound turns on bright emissions with ca. 27-fold fluorescence enhancement, as TICT formation is inhibited in molecular aggregates.

Revealing the switching mechanisms of an off-on-off fluorescent logic gate system.

A deep understanding of fluorescence on-off and off-on switching mechanisms is the foundation for rationally designing highly effective molecular logic gate components and systems. These mechanisms, however, are often subtle to perceive and interpret, as multiple effects may contribute to the change of fluorescence signals. Herein, we systematically investigated the 'off-on-off' switching mechanisms of a fluorescent logic gate molecule M1 using density functional theory (DFT) and time-dependent DFT (TD-DFT). Based on photoexcitation and photoemission calculations, and potential energy surface scans in the excited state, we have shown that as the pH of the medium continuously decreases and the sequential protonation of the molecule takes place, the prevention of twisted intramolecular charge transfer (TICT) followed by the activation of photo-induced electron transfer (PET) was responsible for the off-on-off switching mechanism of M1. Our results provided new insights for understanding the 'off-on-off' phenomenon in M1. The good agreement between theoretical calculations and experimental observations also suggests that computational chemistry is a powerful tool to aid the molecular design and engineering of fluorescent logic gate compounds.