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In the realm of cell research, membraneless organelles have become a subject of increasing interest. However, their ever‐changing and amorphous morphological characteristics have long presented a formidable challenge when it comes to studying their structure and function. In this paper, a fluorescent probe Nu‐AN is reported, which exhibits the remarkable capability to selectively bind to and visualize the nucleolus morphology, the largest membraneless organelle within the nucleus. Nu‐AN demonstrates a significant enhancement in fluorescence upon its selective binding to nucleolar RNA, due to the inhibited twisted intramolecular charge–transfer (TICT) and reduced hydrogen bonding with water. What sets Nu‐AN apart is its neutral charge and weak interaction with nucleolus RNA, enabling it to label the nucleolus selectively and reversibly. This not only reduces interference but also permits the replacement of photobleached probes with fresh ones outside the nucleolus, thereby preserving imaging photostability. By closely monitoring morphology‐specific changes in the nucleolus with this buffering fluorogenic probe, screenings for agents are conducted that induce nucleolar stress within living cells. [ABSTRACT FROM AUTHOR]

In this study, we have uncovered that trifluoroethylamine-substituted solvatochromic fluorophores maintain consistently high and stable fluorescence intensity in diverse polar environments, including highly polar and protic solvents. The 1,8-naphthalimide derivatives serve as a buffering fluorogenic indicator for lipid droplet morphology during the fusion process and ratiometric probe for microenvironment polarity based on Halo-tag technology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The escalating health threats posed by bacteria and the pressing issue of antibiotic resistance underscore the urgent need for selective detection technologies and antimicrobial reagents. Fluorescein's biocompatibility renders it the preferred fluorescent dye for in vivo imaging. This study reveals that compounds from the aliphatic chain-derived fluorescein series (BMP-alkyl) not only selectively stain and fluorescently image Gram-positive bacteria but also demonstrate the ability to rapidly eliminate them. The discovery of fluorescein's antibacterial properties is poised to have applications in antibiotic research due to its excellent biocompatibility. These amphiphilic BMP-alkyl compounds were easily synthesized through a one-step amidation process involving dicarboxyfluorescein and long-chain fatty amines. In aqueous solutions, the formed aggregates exhibited complete fluorescence quenching. Upon encountering the Gram-positive bacterial membrane, BMP-alkyl compounds underwent depolymerization, resulting in a single fluorescent molecule and the activation of fluorescence. Subsequently, as these amphiphilic fluoresceins continuously combined and entered the bacterial interior, the potential for bacterial membrane depolarization, membrane structure damage, and ultimately bacterial death ensued. [Display omitted] • Fluorescein was found to be fluorescent antibiotics against Gram-positive bacteria. • Aliphatic chain-derived fluorescein has dual functions of bacterial imaging and antibiotics. • Fluorescein aggregation and depolymerization modulates wash-free imaging of bacteria. • Antibacterial activity results from fluorescein's negative charge altering bacterial membrane potential. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Subcellular pH plays a key role to modulate protein structures or specific active sites to impact diverse biological processes. The heterogeneity and high dynamics of intracellular pH require probes that can detect pH in the vicinity of the protein and have a wide pH response range. Here, we reported a protein proximity ligation fluorescent probe for super-resolution imaging of subcellular pH dynamics. Relying on SNAP-tag protein labeling technology, the pH-sensitive fluorophore BG-NDM based on photo-induced electron transfer mechanism was localized to the adjacent positions of the target protein in different subcellular organelles. The equilibrium between the non-fluorescent aggregate and the fluorogenic monomer enabled BG-NDM to have the ability of wash-free super-resolution imaging in live cells. And the wide pH response ranges from pH 6 to pH 12 made the probe have the ability of recognizing pH dynamics. The pH changes around functional proteins during mitophagy, cellular alkalosis and acidosis were revealed. Through long-term super-resolution imaging, we could further record the pH dynamics around mitochondrial proteins COX8A during mitochondrial contacts, fusion and fission at the resolution of a single mitochondria. [Display omitted] • Probe BG-NDM was used to super-resolution imaging of subcellular pH dynamics. • SNAP-tag labeling ensures that BG-NDM detects the pH near the protein of interest. • The aggregate/monomer equilibrium endowed BG-NDM wash-free imaging in living cells. • Mitochondrial pH dynamics was recorded with Long-term SIM imaging. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Charge transfer and separation are important processes governing numerous chemical reactions. Fundamental understanding of these processes and the underlying mechanisms is critical for photochemistry. Herein, we report the discovery of a new charge‐transfer and separation process, namely the twisted intramolecular charge shuttle (TICS). In TICS systems, the donor and acceptor moieties dynamically switch roles in the excited state because of an approximately 90° intramolecular rotation. TICS systems thus exhibit charge shuttling. TICSs exist in several chemical families of fluorophores (such as coumarin, BODIPY, and oxygen/carbon/silicon–rhodamine), and could be utilized to construct functional fluorescent probes (i.e. viscosity‐ or biomolecule‐sensing probes). The discovery of the TICS process expands the current perspectives of charge‐transfer processes and will inspire future applications. [ABSTRACT FROM AUTHOR]

Charge transfer and separation are important processes governing numerous chemical reactions. Fundamental understanding of these processes and the underlying mechanisms is critical for photochemistry. Herein, we report the discovery of a new charge‐transfer and separation process, namely the twisted intramolecular charge shuttle (TICS). In TICS systems, the donor and acceptor moieties dynamically switch roles in the excited state because of an approximately 90° intramolecular rotation. TICS systems thus exhibit charge shuttling. TICSs exist in several chemical families of fluorophores (such as coumarin, BODIPY, and oxygen/carbon/silicon–rhodamine), and could be utilized to construct functional fluorescent probes (i.e. viscosity‐ or biomolecule‐sensing probes). The discovery of the TICS process expands the current perspectives of charge‐transfer processes and will inspire future applications. [ABSTRACT FROM AUTHOR]

The imidazolium-pyrene self-assembling nanoprobes can rapidly discriminate four types of surfactants. The surfactants disassembled the probes to turn on significant fluorescence and transform between a pyrene monomer and an excimer, generating a two-dimensional analysis map. The probes were further used to identify different bacterial species. [ABSTRACT FROM AUTHOR]

SNAP-tag is one of most popular genetically encoded protein tags that can be labeled with fluorescent molecules for visualizing a protein of interest in live cells. Fluorogenic probes keep dark until they label protein tags, significantly improving the signal-to-noise ratio to image proteins without wash-out step. However, most of reported fluorogenic probes for SNAP-tag suffered from the low or mild labeling rate comparing with non-fluorogenic ones. In this paper, we reported a 4-amino-naphthliamide derived fluorogenic probe for SNAP-tag, which exhibited the fast record labeling rate among fluorogenic probes. Finally, we applied this probe to image proteins in mitochondria and nucleus in live cells without wash-out steps. [ABSTRACT FROM AUTHOR]

Fluorescent displacement assay has been used as an effective method to screen target protein inhibitors, but there is a lack of reports on the detection of small molecule inhibitor activity by tracking the binding and dissociation of fluorescent substrates to target proteins in living cells. In this paper, we synthesized a cell membrane-impermeable benzenesulfonamide-conjugated rhodamine 6G derivative, CA532 , which dynamically binds to cells expressing carbonic anhydrase IX (CAIX) for fluorescence imaging of cell membranes, while small molecule inhibitors competitively displace CA532 from CAIX, resulting in a decrease in the fluorescence intensity on the cell membrane. The activity of different inhibitors cause concentration-dependent dynamic binding and dissociation of protein-substrate complexes, and the associated diverse replacement processes can be fully revealed by fluorescence imaging of cell membranes, which also enables CA532 to screen CAIX inhibitors in situ in living cells. • A cell membrane-impermeable fluorescent probe for CAIX protein was developed. • CAIX on the cell membrane can be imaged stably and dynamically for a long time • The existence of tertiary amide ligand renders rhodamine complete membrane impermeability. • The activities of CAIX inhibitors would be imaged in-situ. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A 1,8-naphthalimide-derived fluorogenic probe was reported to label SNAP-tag fusion proteins in living cells. The probe can rapidly label a SNAP-tag and exhibit a fluorescence increase of 36-fold due to the additive effects of environment sensitivity of fluorophores and inhibition of photo-induced electron transfer from O6-benzylguanine to the fluorophore. The labeling of intracellular proteins has been successfully achieved without a wash-out procedure. [ABSTRACT FROM AUTHOR]

An NBD-derived fluorescent sensor termed CdTS was reported to sense Cd 2+ with very high binding selectivity and significant fluorescence turn-on signal selectivity (65 fold enhancement). The amide/di-2-picolylamine receptor binds Cd 2+ in an imidic acid tautomeric form, but binds most of other metal ions in an amide tautomeric form. The transformable ability makes CdTS have the specific selectivity for Cd 2+ . Additionally, CdTS can fluoresently and colorimetricly recognize Cd 2+ across a wide pH range from 4.5 to 11.5. Finally, we applied CdTS to detect Cd 2+ in living cells. [ABSTRACT FROM AUTHOR]

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying mitochondrial dynamics in living cells. However, the lack of photostable and chemstable probe makes long-term super-resolution imaging of mitochondria still a challenging work. Herein, we reported a 4-azetidinyl-naphthliamide derived SNAP-tag probe AN-BG exhibiting excellent fluorogenicity and photostability for long-term super-resolution imaging of mitochondrial dynamics. The azetidinyl group and naphthalimide fluorophore are in a flat conformation which can effectively suppress twisted intramolecular charge transfer and then effectively improve the brightness and photostability. This planarized molecular structure is conducive to the formation of fluorescence-quenched J -aggregates, and the protein labeling process will depolymerize the probes and restore fluorescence. Fluorescent labeling mitochondrial inner membrane proteins via SNAP tags overcomes the shortcomings that variations in mitochondrial inner membrane potential will release probes attached to mitochondria by electrostatic interactions. Therefore, AN-BG realized the stable labeling of mitochondria and the long-term imaging of mitochondrial dynamics under super-resolution microscopy. Image 1 • A naphthliamide-derived fluorogenic probe for SNAP-tag was developed. • Fluorescent labeling of mitochondria was achieved via SNAP-tag fused proteins. • Long-term super-resolution imaging of mitochondrial dynamics was achieved. • The dynamic mitochondrial fusion and fission were imaged with fluorescent nanoscopy. • Two-color SIM imaging revealed multiple modes of action between mitochondria and lysosomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We reported fluorescent probes to image Zn2+ with plasma membrane-specific and Zn2+-specific fluorogenicities. The probes contained hydrophobic alkyl chains as membrane-anchored domains and hydrophilic zinc sensor ZTRS, and aggregated to display quenched fluorescence. Cells dissolved the aggregates and the liberated probes were dispersed on the outside of the cell plasma membrane. Aggregates that did not bind to the cell membrane still exhibited aggregation-induced fluorescence quenching after complexing with zinc ions, while probes anchored on the membrane surface exhibited a fluorescence-enhanced response upon recognition of zinc ions. [ABSTRACT FROM AUTHOR]

We have, for the first time, reported a fluorescent probe (ZTRS–C18–Cd(ii) complex) which discriminated four types of surfactants. This recognition was realized depending on the transformation of ZTRS–Cd2+ binding patterns in different microenvironments formed in various types of surfactants. [ABSTRACT FROM AUTHOR]

A fluorescent sensor for halogenated solvents termed AMN is reported. AMN shows strong fluorescence in most halogenated solvents (QE ＞ 0.1) but weak fluorescence (QE＜0.01) in most non-halogenated solvents. In chlorinated solvents, the fluorescence intensity decreased with the reduction of chlorine content. On the contrary, in brominated solvents the fluorescence intensity increased with the reduction of bromine content. It is worth mentioning that AMN displayed fluorescence emission centered at 520 nm in CCl4 with a quantum yield of 0.607, at 556 nm in CHCl3 with a quantum yield of 0.318, at 584 nm in CH2Cl2 with a quantum yield of 0.128, whereas in CHBr3 was centered at 441 nm with a quantum yield of 0.012. AMN was shown to have the ability to differentiate CCl4, CHCl3, CH2Cl2 and CHBr3 halogenated solvents. [ABSTRACT FROM AUTHOR]

The emission intensities of coumarin 545 solution exhibit a low temperature dependence, with a record-low temperature coefficient of only ∼0.025% per °C. This monomer-aggregate coupled fluorescence system can be used for ratiometric temperature measurements with high spatial and temporal resolutions; three different working modes have been demonstrated. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Fluorescent probes for hydrogen sulfide have received considerable attention because of the biological significance of H2S recognized recently. Two-photo microscopy offers advantages of increased penetration depth, localized excitation, and prolonged observation time. However, two-photon fluorescent probes for H2S are still rare. In this work, we introduced a dinitrophenyl ether group into the 4-position of 1,8-naphthalimide, which acts as the H2S reactive site, to efficiently yield compound NI–NHS as a two-photo fluorescent probe for H2S. The probe NI–NHS has a high selectivity for H2S over competitive anions and sulfide-containing analytes. This probe exhibits turn-on fluorescence detection of H2S in bovine serum and two-photon fluorescent imaging of H2S in living cells. [Copyright &y& Elsevier]

Abstract: Fluorescent probes for hydrogen sulfide have received considerable attention because of the biological significance of H2S recognized recently. However, near-infrared fluorescent probes for H2S are still rare. In this work, a new near-infrared fluorescent probe for H2S was developed based on the reduction reaction of azide with H2S to amine based on the fluorophore of dicyanomethylene-4H-chromene because of its long excitation and emission wavelength. The probe has a high selectivity for H2S over competitive anions and sulfide-containing analytes. Finally, the probe was applied to sense H2S in living cells. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Cell surface proteins are responsible for many critical functions. Systematical profiling of these proteins would provide a unique molecular fingerprint to classify cells and provide important information to guide immunotherapy. Cell surface biotinylation method is one of the effective methods for cell surface proteome profiling. However, classical workflows suffer the disadvantage of poor sensitivity. In this work, we presented an optimized protocol which enabled identification of more cell surface proteins from a smaller number of cells. When this protocol was combined with a tip based fractionation scheme, 4510 proteins, including 2055 annotated cell surface-associated proteins, were identified with only 20 microgram protein digest, showing the superior sensitivity of the approach. To enable process 10 times fewer cells, a pipet tip based protocol was developed, which led to the identification of about 600 cell surface-associated proteins. Finally, the new protocol was applied to compare the cell surface proteomes of two breast cancer cell lines, BT474 and MCF7. It was found that many cell surface-associated proteins were differentially expressed. The new protocols were demonstrated to be easy to perform, time-saving, and yielding good selectivity and high sensitivity. We expect this protocol would have broad applications in the future. Significance Cell surface proteins confer specific cellular functions and are easily accessible. They are often used as drug targets and potential biomarkers for prognostic or diagnostic purposes. Thus, efficient methods for profiling cell surface proteins are highly demanded. Cell surface biotinylation method is one of the effective methods for cell surface proteome profiling. However, classical workflows suffer the disadvantage of poor sensitivity. In this work, we presented an optimized protocol which enabled identification of more cell surface proteins from a smaller number of starting cells. The new protocol is easier to perform, time-saving and has less protein loss. By using a special pipet tip, sensitive and in-depth cell surface proteome analysis could be achieved. In combination with label-free quantitative MS, the new protocol can be applied to the differential analysis of the cell surface proteomes between different cell lines to find genetically- or drug-induced changes. We expect this protocol would have broad application in cell surface protein studies, including the discovery of diagnostic marker proteins and potential therapeutic targets. Graphical abstract Unlabelled Image Highlights • An optimized protocol of biotinylation method was developed to identify cell surface proteins. • The new protocol is easier to perform, time-saving and has less protein loss. • By using a special pipet tip, sensitive and in-depth cell surface proteome analysis could be achieved. • About 600 cell surface-associated proteins were identified from only 1 × 105 cells. • The cell surface proteomes of two breast cancer cell lines, BT474 and MCF7, were revealed. [ABSTRACT FROM AUTHOR]

Rhodamine B-naphthalimide (RhB-Naph) demonstrated a distinct aggregation-induced emission (AIE) mechanism, different from the restriction of intramolecular rotations or vibrations as in traditional AIE molecules. The monomers of RhB-Naph were non-emissive, due to the presence of a dark S1 state. Upon molecular aggregation, intermolecular interactions significantly altered the electronic properties of RhB-Naph, leading to the formation of a bright S1 state and endowing RhB-Naph with notable AIE properties. Besides, we demonstrated that RhB-Naph enabled the development of a solid-state three-color fluorescent switch upon multi-external stimuli. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A nonuniform coke location in micrometer-sized SAPO-34 crystals is presented. • Pore blockage by coke is the main contribution to catalyst deactivation. • Coke formation inside cages imposed strong limitations on molecular diffusion. • Coke located at the rims of crystals led to the inaccessibility of inner acid sites. Abstract The whole reaction course of methanol-to-olefins (MTO) conversion over SAPO-34 catalyst with crystal size 7–10 µm has been investigated by multiple spectroscopic techniques. The reaction and deactivation of methanol conversion are closely correlated to the amount, size, and location of coke formed in the CHA nanocages. A nonuniform spatial distribution of these coke species in the individual SAPO-34 crystals is revealed by a combination of hyperpolarized (HP) 129Xe NMR and confocal fluorescence microscopy (CFM) techniques. Combined with acidic properties and diffusion performance, the bulky coke species formed at the shell layer of the crystals led to an increase of intracrystalline diffusion resistance and a decrease in accessible acidic sites in the deactivated catalyst. The overall catalyst deactivation mode of the MTO reaction is presented in terms of coke formation and location, mass transport variation, and accessibility of Brønsted acid sites in the SAPO-34 catalyst. [ABSTRACT FROM AUTHOR]

Genetically encoded light-up RNA aptamers have become powerful tools for visualizing RNAs in cells. However, fluorogenic dye/aptamer pairs used for super-resolution RNA imaging are still rare. Herein, we reported an environment-sensitive dye Nap which can light up its target RNA aptamer NapRA for aptamer-based RNA imaging with confocal and structured illumination microscopy (SIM). Nap was designed to be amphiphilic to assemble into nano-particles in water associated with the quenched fluorescence by the aggregation effects. NapRA was obtained from SELEX technology with a 12-round Nap -based selection and has a high binding affinity to Nap of K d = 3.142 nM. The super affinity disintegrated the dye aggregate and resulted in a turn-on fluorescent signal. The light-up feature combined with high photostability made Nap / NapRA a useful system capable of super-resolution imaging of the expression of RNA in bacteria. This work represents the first application of dye aggregation-disaggregation for aptamer-based RNA imaging. [Display omitted] • A novel fluorogenic dye-aptamer pair was developed for RNA imaging. • Aggregation-disaggregation property of dye Nap was used to light up aptamer. • Aptamers targeting Nap of K d = 3.142 nM was screened by SELEX. • Super-resolution imaging of bacteria mRNA was achieved. [ABSTRACT FROM AUTHOR]

It is urgent to understand the infection mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for the prevention and treatment of COVID-19. The infection of SARS-CoV-2 starts when the receptor-binding domain (RBD) of viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) of the host cell, but the endocytosis details after this binding are not clear. Here, RBD and ACE2 were genetically coded and labeled with organic dyes to track RBD endocytosis in living cells. The photostable dyes enable long-term structured illumination microscopy (SIM) imaging and to quantify RBD-ACE2 binding (RAB) by the intensity ratio of RBD/ACE2 fluorescence. We resolved RAB endocytosis in living cells, including RBD-ACE2 recognition, cofactor-regulated membrane internalization, RAB-bearing vesicle formation and transport, RAB degradation, and downregulation of ACE2. The RAB was found to activate the RBD internalization. After vesicles were transported and matured within cells, RAB was finally degraded after being taken up by lysosomes. This strategy is a promising tool to understand the infection mechanism of SARS-CoV-2. [Display omitted] • RBD endocytosis is resolved by imaging the location and ratio of ACE2/RBD fluorescence • Exploring the initiation and influencing factors of RBD internalization • The movement and maturation of ACE2/RBD co-localized vesicles are tracked by SIM imaging • The RBD-ACE2 complex is taken up and degraded by lysosomes Miao et al. genetically encoded RBD and ACE2 with Halo/SNAP tags and then labeled them with rhodamine/cyanine dyes. SIM imaging resolved endocytosis of RBD-ACE2 binding in living cells, including RBD-ACE2 recognition on the plasma membrane, cofactor-regulated membrane internalization, RAB-bearing vesicle formation and transport, RAB degradation, and downregulation of ACE2. [ABSTRACT FROM AUTHOR]

A simple strategy has been developed to construct high-ordered protein nanotubes using electrostatic interactions and “zero-length” crosslinking induced by small molecular ethylenediamine. Furthermore, utilizing covalent crosslinking, we constructed stable nanoenzymes with multi-glutathione peroxidase (GPx) active centers on the surface of the nanotubes, which were anticipated to be ideal functional bionanomaterials. [ABSTRACT FROM AUTHOR]

The dye rhodamine, as the most popular scaffold to construct fluorescent labels and probes, has been explored extensively on its structure-fluorescence relationships. Particularly, the replacement of the oxygen atom in the 10th position with heteroatoms obtained various new rhodamines with improved photophysical properties, such as brightness, photostability, red-shifted emission and fluorogenicity. However, the applications of heteroatom-substituted rhodamines have been hindered by difficult synthetic routes. Herein, we explored the condensation strategy of diaryl ether analogues and o -tolualdehyde to synthesize various heteroatom-substituted rhodamines. We found that the electron property and steric effect in the rhodamine 10th position determined the synthetic yield. It's concluded that this condensation method was more suitable for the synthesis of heteroatom-substituted rhodamines with small or electron-donating groups like rhodamine, S-rhodamine and Si-rhodamine. We hope these results will benefit the design and synthesis of heteroatom-substituted rhodamines. Unlabelled Image • Heteroatom-substituted rhodamines through the condensation of diaryl ether analogues and o -tolualdehyde were synthesized. • Electron-donating moiety increased the nucleophilic ability of diaryl ether analogues and facilitated the reaction. • Rhodamines with strong electron-withdrawing moiety in the 10th position hindered the reaction and resulted poor yield. [ABSTRACT FROM AUTHOR]