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.

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.

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.

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.

Electrostatically stabilized aggregate(ESAg)——Ⅲ.Evidence derived from the coaggregating behavior of fluorescence probes(FP)with ionic surfactants

Hydr hobie一lipophilic interactions(HLI)will start to bri about the formationof simple aggregates(Ag,s)and eoa egates(CoAg,s)from neutral organie moleeuleswhich possess at least onefl ble chain with more than seven eHZ grou ,1,2 at theeritieala egate eoncentration

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.

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.

Synthesis of 3-hydroxyflavone fluorescent probes and study of their fluorescence properties

Direct measurement of dipole potential in biological membranes has been impossible and 3-hydroxyflavones(3HFs) have allowed detection of changes in dipole potential in biological systems.In the present study,sixteen derivatives of 3HF with aliphatic hydrocarbon chains of different lengths at 4′-position and 6-position were synthesized.The basic fluorescence properties of 3HFs are maintained in all the probes in terms of strong blue shift in maximum fluorescence emission wavelength and>100 fold increase in quantum yield in organic solvents and in dioleoylphosphatidylcholine(DOPC) small unilamellar vesicles(SUV) in comparison to in aqueous Hepes buffer(15 mmol/L,pH 7.4).More importantly,the ability of the new compounds to report dipole potential changes in biological systems are also maintained,since all the new probes showed spectrum properties that are similar to yet different from that of F4N1,which potentially may allow more sensitive measurement of the dipole potential change in membranes.

Activatable fluorescent probes for imaging and diagnosis of rheumatoid arthritis

Rheumatoid arthritis(RA)is a systemic autoimmune disease that is primarily manifested as synovitis and polyarticular opacity and typically leads to serious joint damage and irreversible disability,thus adversely affecting locomotion ability and life quality.Consequently,good prognosis heavily relies on the early diagnosis and effective therapeutic monitoring of RA.Activatable fluorescent probes play vital roles in the detection and imaging of biomarkers for disease diagnosis and in vivo imaging.Herein,we review the fluorescent probes developed for the detection and imaging of RA biomarkers,namely reactive oxygen/nitrogen species(hypochlorous acid,peroxynitrite,hydroxyl radical,nitroxyl),pH,and cysteine,and address the related challenges and prospects to inspire the design of novel fluorescent probes and the improvement of their performance in RA studies.

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.

Hydroxylated graphene quantum dots as fluorescent probes for sensitive detection of metal ions

Highly sensitive methods are important for monitoring the concentration of metal ions in industrial wastewater.Here,we developed a new probe for the determination of metal ions by fluorescence quenching.The probe consists of hydroxylated graphene quantum dots(H-GQDs),prepared from GQDs by electrochemical method followed by surface hydroxylation.It is a non-reactive indicator with high sensitivity and detection limits of 0.01μM for Cu2+,0.005μM for Al3+,0.04μM for Fe3+,and 0.02μM for Cr3+.In addition,the low biotoxicity and excellent solubility of H-GQDs make them promising for application in wastewater metal ion detection.

Determination of Formation Constants of Ce^(3+) Complexes by Fluorescence

Fluorescence of Ce 3+ (aq) is very strong and thoroughly quenched when Ce 3+ forms complexes with ligands containing carboxylate or phosphate groups. This property is employed to develop a new simple method to determine formation constants of Ce 3+ complex system. Fluorescence intensity is used to determine concentration of Ce 3+ (aq) in complex solution, as it is proportional to the concentration of Ce 3+ (aq). In this paper, two examples, Ce 3+ malonic acid and Ce 3+ AMP(adenosine 5′ monophosphate), of fluorescence titration were made and association constants and association numbers of the two systems were deduced for the titration with Scatchard plot. The results are similar to those obtained by others. It is suggested that this Ce 3+ method is a promising fluorescence probe, as it is not only simple but also superior to other lanthanide fluorescence probe in being able to study a large amount of organic ligands and biomolecules.

Computational design of ratiometric two-photon fluorescent Zn2+probes based on quinoline and di-2-picolylamine moieties

To improve two-photon absorption(TPA)response of a newly synthesized probe,a series of ratiometric two-photon fluorescent Zn^(2+) sensors based on quinoline and DPA moieties have been designed.The one-photon absorption,TPA,and emission properties of the experimental and designed probes before and after coordination with Zn^(2+) are investigated employing the density functional theory in combination with response functions.The design consists of two levels.In the first level of design,five probes are constructed through using several electron acceptors or donors to increase accepting or donating ability of the fluorophores.It shows that all the designed probes have stronger TPA intensities at longer wavelengths with respect to the experimental probe because of the increased intra-molecular charge transfer.Moreover,it is found that the probe 4 built by adding an acyl unit has the largest TPA cross section among the designed structures due to the form of longer conjugated length and more linear backbone.One dimethylamino terminal attached along the skeleton can improve TPA intensity more efficiently than two side amino groups.Therefore,in the second level of design,a new probe 7 is formed by both an acyl unit and a dimethylamino terminal.It exhibits that the TPA cross sections of probe 7 and its zinc complex increase dramatically.Furthermore,the fluorescence quantum yields of the designed probes4 and 7 are calculated in a new way,which makes use of the relation between the computed difference of dipole moment and the measured fluorescence quantum yield.The result shows that our design also improves the fluorescence quantum yield considerably.All in all,the designed probes 4 and 7 not only possess enhanced TPA intensities but also have large differences of emission wavelength upon Zn^(2+) coordination and strong fluorescence intensity,which demonstrates that they are potential ratiometric two-photon fluorescent probes.

The LB Films of Dansyl Chloride Labeled Octadecylamine and Its Fluorescence Lifetime

Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) In order to simplify and understand the LB films of fluorescent probe labeling proteins. its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique. Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.

Study on Anticoagulation Factor I from Agkistrodon Acutus Venom by Rare Earth Ion Probes

It was observed that rare earth ions (Nd 3+, Sm 3+, Eu 3+, Gd 3+, Tb 3+) have significant quenching effects on the fluorescence of anticoagulation factor I (ACF I). The results of the fluorescence titration of ACF I with rare earth ions demonstrate that ACF I has two RE 3+-binding sites, and the rare earth ions and Ca 2+ bind to ACF I competitively in the two similar sites. The association constants K 1 and K 2 of ACF I with each rare earth ions (Nd 3+, Sm 3+, Eu 3+, Gd 3+, Tb 3+) are close to each other, which indicates the structural similarity of the two binding sites in ACF I. Although the ionic radii of Nd 3+, Sm 3+, Eu 3+, Gd 3+ and Tb 3+ are different, both their K 1 and K 2 are similar, respectively. This reveals the conformational flexibility of the two binding sites in ACF I, which offers a possibility for Ca 2+ to take play in the inducing conformational changes of ACF I and the promoting the binding of ACF I with activated coagulation factor X.

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.

Ester-Derivatized Indoles as Fluorescent and Infrared Probes for Hydration Environments

Tryptophan derivatives have long been used as site-specific biological probes. 4-Cyanotrypto- phan emits in the visible region and is the smallest blue fluorescent amino acid probe for bio- logical applications. Other indole or tryptophan analogs may emit at even longer wavelengths than 4-cyanotryptophan. We performed FTIR, UV-Vis, and steady-state and time-resolved fluorescence spectroscopy on six ester-derivatized indoles in different solvents. Methyl indole- 4-carboxylate emits at 450 nrn with a long fluorescence lifetirne, and is a promising candidate for a fluorescent probe. The ester-derivatized indoles could be used as spectroscopic probes to study local protein environments. Our measurements provide a guide for choosing esterderivatized indoles to use in practice and data for computational modeling of the effect of substitution on the electronic transitions of indole.

STRUCTURAL INVESTIGATION OF A NEW POLYORGANOSILOXANE CONTAINING NANOTUBULAR CAVITIES USING ORGANIC FLUORESCENT PROBES

Fluorescent molecules - p-dimethylaminobenzonitrile (DMABN) and trans-stilbene (TSB ) - were used as probes to investigate the inner structure of the nanotubular cavities contained in a new polyorganosiloxane (POS) which was prepared by the hydrosilylation coupling reaction of cis-isotactic ladderlike polyvinylsilsesquioxane (Vi-T) with 1,1,3,3-tetramethyldisiloxane (H-MM) as coupling agent in the presence of catalyst dicyclopentadienyldichloroplatinum (Cp2PtCl2). The results from FTIR spectra and fluorescence spectra in combination with molecular simulation reveal that the cross-section of the nanotubular cavity is nearly rectangular in shape, and is about 0.62 nm in width and about 0.38 nm in height. This work gains in-depth knowledge into the inner structure of the nanotubular cavities of POS and furthermore provides a guide to the selection of proper guest molecules in constructing functional supramolecular clathrate based upon POS.

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.