Application and development of fluorescence probes in MINFLUX nanoscopy(invited paper)

The MINimal emission FLUXes(MINFLUX)technique in optical microscopy,widely recognized as the next innovative fluorescence microscopy method,claims a spatial resolution of 1-3 nm in both dead and living cells.To make use of the full resolution of the MINFLUX microscope,it is important to select appropriate fluorescence probes and labeling strategies,especially in living-cell imaging.This paper mainly focuses on recent applications and developments of fluorescence probes and the relevant labeling strategy for MINFLUX microscopy.Moreover,we discuss the deficiencies that need to be addressed in the future and a plan for the possible progression of MINFLUX to help investigators who have been involved in or are just starting in the field of super-resolution imaging microscopy with theoretical support.

A KIND OF FLUORESCENCE PROBE TO STUDY THE KINETICS OF POLYMERIZATION PROCESS

Fluorescence properties of 1-pheny1-3-(4'-nitrophenyl) pyrazoline (PNP) were studied inbulk polymerization process of methylmethacrylate (MMA). The fluorescence intensity of PNPwas enhanced and the emission maximum was blue shifted with the polymerization progress. Inthe period of auto-acceleration of the polymerization the enhancement of fluorescence intensityand blue shift of peak wavelength in spectra could be observed evidently. This means that thesolvatochromic properties of PNP are influenced not only by the solvent polarity but also by theviscosity of the medium (especially by the phase transitiott). In solid state PNP emits from thecharge transfer excited state without solvent relaxation. The transient emission spectra and theresults from Bakhshiev model of solvent relaxation coincide with that from the polymerizationexperiment.

Primary amine coupling on nanocarbon catalysts: Reaction mechanism and kinetics via fluorescence probe analysis

Non-metallic nanocarbon materials catalyzed coupling reactions of primary amines to produce imine is an efficient,green and sustainable synthetic route,which has a wide application prospect in fine chemicals or pharmaceutical molecules.In the present study,we show firstly the relatively high catalytic activity of graphene oxide in the reaction of oxidative coupling of benzylamine(OCB),which is even comparable with typical metal-based catalysts,indicating the great potential of nanocarbon materials in this reaction system.More importantly,a novel twophoton fluorescence probe molecule(N-propyl-4-hydrazinyl-1,8-naphthalimide,NA)with special chemical structure of hydrazine functionality was synthesized.The probe NA could selectively react with aldehyde or ketone compounds,leading to the photoluminescence enhancement via inhibition of photo induced electron transfer(PET)process.The synthesized NA was applied as probe in carbon catalyzed OCB system to predict the existence of reaction intermediate benzaldehyde(BA),indicating the reaction pathway of oxidation-deamination-condensation in nanocarbon catalyzed OCB process.The proposed luminescence-probe strategy for revealing the kinetics and mechanism may also shed light in other reaction systems concerning the intermediates or products of ketones or aldehydes.

SPECTROSCOPIC DETERMINATION OF THE AVERAGE NUMBER OF COAGGREGATES (Nco) OF CHOLESTERYL ESTERS AND THE FLUORESCENCE PROBE

Average aggregate number of coaggregates(N_co)of CE-n or BL-n and the fluoresc- ence probe(Np-16)have been determined by using time-resolved fluorescence spectroscopy. Chain-length,hydroxy-group and chain-foldability effects on the N_co have been discussed.

Sign Response Mechanism of TCA Self-assembled Fluorescence Probe for Cu^2＋ Detection： FRET Evidence by DFT

A new type of self-assembled molecule ON-OFF fluorescence probe for toxic transition metal ions, made up of thiacalix[4]arene, micelle and fluorescence group, has been studied by DFT/TDDFT method combined with experiment spectra. Since the mechanism of the optical quenching signal response of such self-assembled micelle probe has always been a controversial issue of uncertainty, the spatial construction and geometric structures of the functional units of probe in the Cu2＋ ion detecting process were calculated and the mechanism was investigated by the molecular transition orbital pairs method to explore the origination of ON-OFF fluorescence sign response. The results presented that the signal response mechanism of the micelle probe is ascribed to F？rster resonance energy transfer（FRET） which provides new sights different from most of the conclusions by the related research work reported.

An insight into the in vivo imaging potential of curcumin analogues as fluorescence probes

Curcumin and its derivatives have good electrical and optical properties due to the highly symmetric structure of delocalized π electrons. Apart from that, curcumin and its derivatives can interact with numerous molecular targets, thereby exerting less side effects on human body. The fluorescence emission wavelength and fluorescence intensity of curcumin can be enhanced by modifying its π-conjugated system and β-diketone structure. Some curcumin-based fluorescent probes have been utilized to detect soluble/insoluble amyloid-β protein, intracranial reactive oxygen species, cysteine, cancer cells, etc. Based on the binding characteristics of curcumin-based fluorescent probes with various target molecules, the factors affecting the fluorescence intensity and emission wavelength of the probes are analyzed, in order to obtain a curcumin probe with higher sensitivity and selectivity. Such an approach will be greatly applicable to in vivo fluorescence imaging.

A Novel Fluorescence Probe 9-(4-(1,2-diamine)benzeneN^1-phenyl)acridine for Nitric Oxide Determination

A novel fluorescent probe 9-（4-（1,2-diamine）benzene-N1-phenyl）acridine（DABPA） was synthesized for the detection of nitric oxide（NO） and characterized by IR, 1H-NMR and EI-MS spectroscopy. Based on a photoelectron transfer mechanism, the fl uorescence intensities of DABPA were investigated with the different concentrations of NO. Under the optimal experimental conditions, the fl uorescence intensity of DABPA had a good linear relationship（R2=0.9977） with NO concentration in the range from 1×10-7 to 1.5×10-6 mol/L with a detection limit of 1×10-8 mol/L. The cytotoxicity induced by DABPA was evaluated by the MTT（3-（4,5-dimethylthiazol-2-yl）-2,5diphenyl tetrazolium bromide） assay for biological application. Furthermore, the probe DABPA had also been successfully applied to real-time image NO produced in PC12 cells in the presence of L-arginine.

Lighting up plants with near-infrared fluorescence probes

Fluorescence imaging is a non-invasive and highly sensitive bioimaging technique that has shown remarkable strides in plant science. It enables real-time monitoring and analysis of biological and pathological processes in plants by labeling specific molecular or cellular structures with fluorescent probes. However, tissue scattering and phytochrome interference have been obstacles for conventional fluorescence imaging of plants in the ultraviolet and visible spectrum, resulting in unsatisfactory imaging quality. Fortunately, advances in near-infrared(NIR) fluorescence imaging technology(650-900 nm) offer superior spatial-temporal resolution and reduced tissue scattering, which is sure to improve plant imaging quality. In this review, we summarize recent progress in the development of NIR fluorescence imaging probes and their applications for in vivo plant imaging and the identification of plant-related biomolecules. We hope this review provides a new perspective for plant science research and highlights NIR fluorescence imaging as a powerful tool for analyzing plant physiology, adaptive mechanisms, and coping with environmental stress in the near future.

A ratiometric fluorescent probe for hypoxanthine detection in aquatic products based on the enzyme mimics and fluorescence of cobalt-doped carbon nitride

A ratiometric fluorescent probe for hypoxanthine(Hx)detection was established based on the mimic enzyme and fluorescence characteristics of cobalt-doped graphite-phase carbon nitride(Co doped g-C_(3)N_(4)).In addition to emitting strong fluorescence,the peroxidase activity of Co doped g-C_(3)N_(4)can catalyze the reaction of O-phenylenediamine and H_(2)O_(2)to produce diallyl phthalate which can emit yellow fluorescence at 570 nm.Through the decomposition of Hx by xanthine oxidase,Hx can be indirectly detected by the generating hydrogen peroxide based on the measurement of fluorescent ratio I(F_(570)/F_(370)).The linear range was 1.7-272.2 mg/kg(R^(2)=0.997),and the detection limit was 1.52 mg/kg(3σ/K,n=9).The established method was applied to Hx detection in bass,grass carp,and shrimp,and the data were verified by HPLC.The result shows that the established probe is sensitive,accurate,and reliable,and can be used for Hx detection in aquatic products.

A 4-aminonaphthalimide based environmentally sensitive fluorescence probe

A simply synthesized 4-aminonaphthalimide derivative 1 expresses both polarity and viscosity sensitive fluorescence spectra,indicating its potential usage as an environmentally sensitive fluorescence probe. By comparing the fluorescence behavior of 1 with that of a known 4-aminonaphthalimide derivative 2,it was found that the substitution of the 4-amino group has profound influence on the environmentally sensitive fluorescence properties of 4-aminonaphthalimide.

A ratiometric benzothiazole-based fluorescence probe for selectively recognizing HClO and its practical applications

A ratiometric probe(HBT-HBZ)bearing 2-hydrazino benzothiazole and 3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzaldehyde for sensing hypochlorous acid(HClO)with high selectivity and sensitivity is reported in this article.The fluorescence intensity ratios(I470nm/I572nm)of the probe with different concentrations of analyte showed excellent selectivity and a linear response to minor changes in HClO.The detection limit of 24 nmol/L suggests that the sensor is very sensitive to HClO.According to the series of performed experiments,HBT-HBZ has practical applications,such as the detection of HClO residues in tap water,which has been rarely reported.In addition,confocal laser microscopy experiments confirmed that HBT-HBZ can selectively recognize HClO in HeLa cells.A ratiometric probe(HBT-HBZ)for sensing HClO with high selectivity and sensitivity is reported in this article.The probe exhibited high selectivity for HClO among other ROS,RNS and anions.In addition,HBTHBZ has some practical applications such as the analysis of the HClO content in tap water.Furthermore,confocal fluorescence microscopy imaging showed that HBT-HBZ can be applied for detecting HClO in living cells.

In-situ hydrophobic environment triggering reactive fluorescence probe to real-time monitor mitochondrial DNA damage

Mitochondrial DNA has a special structure that is prone to damage resulting in many serious diseases,such as genetic diseases and cancers.Therefore,the rapid and specific monitoring of mitochondrial DNA damage is urgently needed for biological recognition.Herein,we constructed an in situ hydrophobic environment-triggering reactive fluorescence probe named MBI-CN.The fluorophore was 2-styrene-1H-benzo[d]imidazole,and malononitrile was introduced as a core into a molecule to initiate the hydrolysis reaction in the specific environment containing damaged mitochondrial DNA.In this design,MBI-CN conjugates to mitochondrial DNA without causing additional damages.Thus,MBI-CN can be hydrolyzed to generate MBI-CHO in an in situ hydrophobic environment with mitochondrial DNA damage.Meanwhile,MBI-CHO immediately emitted a significative fluorescence signal changes at 437 and 553 nm within 25 s for the damaged mitochondria DNA.Give that the specific and rapid response of MBI-CN does not cause additional damages to mitochondrial DNA,it is a potentially effective detection tool for the real-time monitoring of mitochondrial DNA damage during cell apoptosis and initial assessment of cell apoptosis.

Fine-tailoring the linker of near-infrared fluorescence probes for nitroreductase imaging in hypoxic tumor cells

Imaging hypoxia using fluorescence probes for nitroreductase（NTR） have attracted much attention in last decade. At least three different linkers have been commonly used to connect the recognition unit and reporting unit in reported probes for NTR. Meanwhile, the linker is known to be a key factor for achieving best sensing performance. In this work, three near-infrared fluorescence probes CyNP-1, CyNP-2 and CyNP-3 were designed and synthesized from an aminocyanine dye CyNP. The three probes have the same recognition unit and same fluorescence reporting unit, but different linkers. CyNP-1 was found to have the best sensing performance for NTR with 40-fold of fluorescence enhancement. It is well investigated how the difference of the linkers brings out the different sensing performance by HPLC, MS and docking calculations. In the end, CyNP-1 was found to have good selectivity for NTR and used to imaging hypoxia in Hela cells.

Highly selective and turn-on fluorescence probe with red shift emission for naked-eye detecting Al^(3+)and Ga^(3+)based on metal-organic framework

A novel ZnII-based metal-organic framework with the formula of{[Zn_(2)(BBIP)_(2)(NDC)_(2)]·H_(2)O}n(JXUST-5)derived from 3,5-bis(benzimidazol-1-yl)pyridine(BBIP)and 1,4-naphthalenedicarboxylic acid(H_(2)NDC)has been synthesized.The adjacent Zn^(II)ions are linked through two BBIP ligands to form a[Zn_(2)(BBIP)_(2)]secondary building unit(SBU).The neighbouring SBUs are further connected by NDC^(2-)withμ2-η^(1):η^(1)andμ2-η^(1):η^(1):η^(1)bridging modes to form a two-dimensional(2D)framework.Topological analysis shows that JXUST-5 could be simplified as an uninodal fes topology with a point symbol of{4.8^(2)}.Furthermore,the 2D framework net could be extended through C-H···πinteraction to form the three-dimensional supramolecular structure.Luminescent experiments suggest that JXUST-5 could selectively and sensitively recognize Al^(3+)and Ga^(3+)through fluorescence enhancement effect along with a relatively large red shift.The detection limits for Al^(3+)and Ga^(3+)are 0.17 and 0.69 ppm,respectively.Interestingly,the sensing process for both Al^(3+)and Ga^(3+)could be directly observed with naked eyes under 365 nm UV lamp.Notably,JXUST-5 could be recycled at least five times as a fluorescent sensor toward Al^(3+)and Ga^(3+),which is the second example of turn-on MOF based fluorescent sensor toward Ga^(3+).

Activatable molecular fluorescence probes for the imaging and detection of ischemic stroke

The real-time, noninvasive, nonionizing, high spatiotemporal resolution, and flexibility characteristics of molecular fluorescence imaging provide a uniquely powerful approach to imaging and monitoring the physiology and pathophysiology of ischemic stroke. Currently, various fluorescence probes have been synthesized with the aim of improving quantitative and quantitative studies of the pathologic processes of ischemic stroke in living animals. In this review, we present an overview of current activatable fluorescence probes for the imaging and diagnosis of ischemic stroke in animal models. We categorize the probes based on their activatable signals from the biomarkers associated with ischemic stroke, and we present representative examples of their functional mechanisms. Finally, we briefly discuss future perspectives in this field.

Rational design of shortwave infrared(SWIR) fluorescence probe:Cooperation of ICT and ESIPT processes for sensing endogenous cysteine

Cysteine is well-known to be an important biothiol and related to many diseases. However, the in vivo detection of endogenous cysteine still suffers from lacking small-molecule fluorophores with both excitation and emission in the near-infrared(650-900 nm)/shortwave-infrared region. Herein, we report a molecular engineering strategy for shortwave infrared(SWIR, 900-1700 nm) sensing of cysteine, which integrated an excited-state intermolecular proton transfer(ESIPT) building block into the intramolecular charge transfer(ICT) scaffold. The obtained novel fluorophore SH-OH displays a maximum absorption at the NIR region, and emission at the SWIR region. We introduce the cysteine-recognition moiety to SH-OH structure, and demonstrate sensing of endogenous cysteine in living animals, using the SWIR emission as a reliable off-on fluorescence signal. This fluorophore design strategy of cooperation of ICT and ESIPT processes expands the in vivo sensing toolbox for accurate analysis in clinical applications.

A Selective and Sensitive Fluorescence Probe for Se（IV） Based on Fluorescence Quenching of Gatifloxacin

A novel high selective and sensitive fluorescence probe termed gatifloxacin was discovered based on fluorescence ＂on-off＂ phenomenon in the presence of Se（IV）. In the Tris-HCl/acetonitrile（3：7, volume ratio, Tris-HCl 0.05 mol/L, pH=7.3） system, the fluorescence intensity of gatifloxacin was linearly decreased with the concentration increase of Se（IV） in a range of 1.0×10-5--5.0×10-5 mol/L with a correlation coefficient of 0.9979（R2=0.9958） and in a range of 5.0×10-5--1.0 ×10-4 mol/L with a correlation coefficient of 0.9973（R2=0.9946）. The detection limit of Se（IV） was 1.70×10-6mol/L.

Response of indigenous Cd-tolerant electrochemically active bacteria in MECs toward exotic Cr（VI） based on the sensing of fluorescence probes

Electrochemically active bacteria （EAB） on the cathodes of microbial electrolysis cells （MECs） can remove metals from the catholyte, but the response of these indigenous EAB toward exotic metals has not been examined, particularly from the perspective of the co-presence of Cd（II） and Cr（VI） in a wastewater. Four known indigenous Cd-tolerant EAB of Ochrobactrum sp X l, Pseudomonas sp X3, Pseudomonas delhiensis X5, and Ochrobactrum anthropi X7 removed more Cd（II） and less Cr（VI） in the simultaneous presence of Cd（II） and Cr（VI）, compared to the controls with individual Cd（II） or single Cr（VI）. Response of these EAB toward exotic Cr（VI） was related to the associated subcellular metal distribution based on the sensing of fluorescence probes. EAB cell membrane harbored more cadmium than chromium and cytoplasm located more chromium than cadmium, among which the imaging ofintracelluler Cr（III） ions increased over time, contrary to the decreased trend for Cd（II） ions. Compared to the controls with single Cd（II）, exotic Cr（VI） decreased the imaging of Cd（II） ions in the EAB at an initial 2 h and negligibly affected therealier. However, Cd（II） diminished the imaging of Cr （III） ions in the EAB over time, compared to the controls with individual Cr（VI）. Current accelerated the harboring of cadmium at an initial 2 h and directed the accumulation of chromium in EAB over time. This study provides a viable approach for simultaneously quantitatively imaging Cd（II） and Cr （III） ions in EAB and thus gives valuable insights into the response of indigenous Cd-tolerant EAB toward exotic Cr（VI） in MECs.

Metal-organic cage as fluorescent probe for LiPF_(6)in lithium batteries

Lithium hexafluorophosphate(LiPF_(6)),the most commonly used lithium battery electrolyte salt,is vulnerable to heat and humidity.Quantitative and qualitative determination the variation of LiPF_(6)have always relied on advanced equipment.Herein,we develop a fast,convenient,high-selective fluorescence detection method based on metal-organic cages(MOC),whose emission is enhanced by nearly 20 times in the presence of LiPF_(6)with good stability and photobleaching resistance.The fluorescent probe can also detect moisture in battery electrolyte.We propose and verify that the luminescence enhancement is due to the presence of hydrogen bond-induced enhanced emission effect in cages.Fluorescent excitation-emission matrix spectra and variable-temperature nuclear magnetic resonance spectroscopy are employed to clarify the role of hydrogen bonds in guest-loaded cages.Density functional theory(DFT)calculation is applied to simulate the structure of host-guest complexes and estimate the adsorption energy involved in the system.The precisely matched lock-and-key model paves a new way for designing and fabricating novel host structures,enabling specific recognition of other target compounds.

A fluorescence probe study of the mixed surfactant vesicles

Vesicle can be prepared from aqueous mixtures of simple commercially available, single-tailed cationic and anionic surfactants. In this work, the I3/I1 value, Ie/Im value, and fluorescence lifetime of pyrene in different systems (see the preparation of samples) were determined. The essential affecting factors in the formation of vesicle can be deduced from the obtained results. It showed that large vesicle must form naturally before sanitation in 0.082 M octyltrimethylammonium bromide and sodium laurate pH=9.2 aqueous solution. While after sonication, only small vesicle exists, which can be proved further through electron microscope.