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There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations of cellular processes by more than one technique. Fluorescent nanodiamond-gold nanoparticles (FND-Au) constitute a new class of "all-in-one" hybrid particles providing unique features for multimodal cellular imaging including optical imaging, electron microscopy, and, and potentially even quantum sensing. Confocal and optical coherence microscopy of the FND-Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because the FND emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) analysis of FND-Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-dimensional cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of FND-Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in subcellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual FND-Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND-Au unique candidates for multimodal optical-electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells. [ABSTRACT FROM AUTHOR]

Aqueous sulfides are emerging signaling agents implicated in various pathological and physiological processes. The development of sensitive and selective methods for the sensing of these sulfides is therefore very important. Herein, we report that the as-synthesized 1-oxo-1H-phenaIene-2,3-dicarbonitrile (OPD) compound provides promising fluorescent properties and unique reactive properties toward aqueous sulfides. It was found that OPD showed high selectivity and sensitivity toward Na2S over thiols and other inorganic sulfur compounds through a sulfide involved reaction which was confirmed by high-resolution mass spectroscopy (HRMS) and nuclear magnetic resonance (NMR) results. The fluorescence intensity increases linearly with sulfide concentration in the range of 1.0-30 μM with a limit of detection of 52 nM. This novel fluorescent probe was further exploited for the fluorescence imaging sensing of aqueous sulfide in HeLa cells. [ABSTRACT FROM AUTHOR]

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules. [ABSTRACT FROM AUTHOR]

A ferroceneunit is introduced into the salicylaldehyde Rhodamine6G hydrazone with an alkynyl group to constitute a new chemosensor, R6G-FC, for specifically sensing Cu2+over other metal ions with multisignals. Upon interaction with Cu2+in aqueous solution, R6G-FCexpressesa detectable fluorescence enhancement along with changes in UV–visabsorption spectra and electrochemical parameters due to the binding-inducedspirolactam ring-opening process in Rhodamine 6G. The complexationprocess between R6G-FCand Cu2+can be carried out within a wide pH range in a short response timeand with a good reversibility. R6G-FCalsoexhibits an excellent performance in sensing Cu2+in the“dip-stick” method with an evident color gradation,and confocal fluorescence microscopy imaging results reveal that R6G-FCcan be used to visualize Cu2+in living HeLa cells with low cytotoxicity. [ABSTRACT FROM AUTHOR]

Achieving highly selective and sensitive detection/visualization of intracellular biological events through the use of cell-penetrable, bioanalyte-activatable, tum-on probes is dependent on the presence of specific event-linked cellular biomarkers, if and only if there exist activatable probes that appropriately respond to the biomarker analyte. Here is described the evaluation of, and use in cellular imaging studies, a previously undisclosed naphthalimide probe QMeNN, whose fluorescence is deactivated by photoinduced electron transfer (PeT) quenching that results from the presence of a covalently linked biomarkerspecific quinone trigger group. Highly selective and rapid activation of the quinone group by the human cancer tumor-linked NAD(P)H:quinone oxido-reductase isozyme 1 (hNQO1) results in fast trigger group removal to yield a highly fluorescent green-energy-range reporter that possesses a high molar absorptivity; there is a 136-fold increase in brightness for the enzymatically produced reporter versus probe precursor, a value 4 times greater than previously reported for the hNQO1 analyte. The novel probe is taken up and activated rapidly within only hNQO1 -positive human cancer cells; addition of an hNQO1 inhibitor prevents the selective activation of the probe. Comparison of cytosolic fluorescence intensity in positive cells versus background in negative cells yields a quantitative metric (positive-to-negative ratio, PNR) for judging hNQO1 activity. We show it is possible to determine hNQOl presence in previously studied colorectal cancer cells and the unexplored ovarian cancer cell line NIH:OVCAR-3, with respective PNR values of 926 and 34 being obtained. Even with 10 min probe incubation, ready discrimination of positive cells from negative cells is achieved. Cell viability is unaffected by probe presence, thereby highlighting the practicality of probe use in live-cell imaging applications. [ABSTRACT FROM AUTHOR]

We fabricated a multifunctional theragnostic agent Ag-Sgc8-FAM for apoptosis-based cancer therapy and fluorescence-enhanced cell imaging. For cancer therapy, aptamers Sgc8 and TDO5 acted as recognizing molecules to bind CCRF-CEM and Ramos cells specifically. It was found that aptamer-silver conjugates (Ag-Sgc8, Ag-TDO5) could be internalized into cells by receptor-mediated endocytosis, inducing specific apoptosis of CCRF-CEM and Ramos cells. The apoptosis of cells depended on the concentration of aptamer-silver conjugates, as well as the incubation time between cells and aptamer-silver conjugates. The apoptotic effects on CCRF-CEM and Ramos cells were different. Annexin V/PI staining, AO/PI staining, MTT assays and ROS (reactive oxygen species) detection demonstrated the specific apoptosis of CCRF-CEM and Ramos cells. For fluorescence-enhanced cell imaging, Ag-Sgc8-FAM was prepared. Compared to Sgc8-FAM molecules, Ag-Sgc8-FAM was an excellent imaging agent as numerous Sgc8-FAM molecules were enriched on the surface of AgNPs for multiple binding with CCRF-CEM cells and signal amplification. Moreover, AgNPs could increase the fluorescence intensity of FAM by metal-enhanced fluorescence (MEF) effect. Therefore, aptamer-silver conjugates can be potential theragnostic agents for inducing specific apoptosis of cells and achieving cells imaging in real time. [ABSTRACT FROM AUTHOR]

Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomel and the development of the lysosomal storage disease known as Gaucher's disease. More recently, mutations in the GBAI gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson's disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within live mammalian cells. Here we describe the successful modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (~99.9%) fluorescent substrates also permit assessment of GCase inhibition in five cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases for use in live cell imaging. [ABSTRACT FROM AUTHOR]

Inthis article, ionic liquid-functionalized carbon nanodots (IL-CDs)were produced in a simple manner by electrochemical exfoliation ofgraphite rods in the presence of an amino-terminated ionic liquid,and their preliminary applications were exploited. TEM and AFM resultsshowed that these IL-CDs are about 2.6 nm in diameter. The small-sizedIL-CDs have strong photoluminescence, with a quantum yield of about11.3%, and could be used for cell imaging. Moreover, the IL-CDs exhibitgood electron transfer properties and catalytic activities for O2and H2O2reduction. Additionally, theas-prepared IL-CDs can be applied as a matrix for immobilizing enzymes(glucose oxidase) to construct biosensors. Due to these favorableproperties, IL-CDs will find promising practical applications in electrocatalysis,biosensing, and bioimaging. [ABSTRACT FROM AUTHOR]

Highly fluorescent N-substituted1-cyanobenz[f]isoindole chitosans (CBI-CSs) withvarious degrees of N-substitution(DS) were synthesized by reacting chitosan (CS) with naphthalene-2,3-dicarboxaldehyde(NDA) in the presence of cyanide under mild acidic conditions. Introductionof 1-cyanobenz[f]isoindole moieties into the CS backboneresulted in lowering of polymer thermal stability and crystallinity.The fluorescence quantum yield (Φf) of CBI-CS wasfound to be DS- and molecular-weight-dependent, with Φfdecreasing as DS and molecular weight were increased. At similarDS values, CBI-CS exhibited 26 times higher Φfincomparison with fluorescein isothiocyanate-substituted chitosan (FITC-CS).CBI-CS/TPP nanoparticles were fabricated using an ionotropic gelationmethod in which pentasodium triphosphate (TPP) acted as a cross-linkingagent. CS and CBI-CS exhibited low cytotoxicity to normal skin fibroblastcells over a concentration range of 0.1–1000 μg/mL, whilean increased cytotoxicity level was evident in CBI-CS/TPP nanoparticlesat concentrations greater than 100 μg/mL. In contrast with CBI-CSpolymers, the CBI-CS/TPP nanoparticles exhibited lower fluorescence;however, confocal microscopy results showed that living normal skinfibroblast cells became fluorescent on nanoparticle uptake. Theseresults suggest that CBI-CS and fabricated nanoparticles thereof maybe promising fluorescence probes for live cell imaging. [ABSTRACT FROM AUTHOR]

A new benzoyl hydrazone based chemosensor R is synthesized by Schiff base condensation of 2,6-diformyl-4-methylphenol and phenyl carbohydrazide and acts as a highly selective fluorescence sensor for Cu2+ and Zn2+ ions in aqueous media. The reaction of R with CuCl2 or ZnCl2 forms the corresponding dimeric dicopper(II) [Cu2(R)(CH3O)(NO3)]2(CH3O)2 (R-Cu2+) and dizinc(II) [Zn2(R)2](NO3)2 (R-Zn2+) complexes, which are characterized, as R, by conventional techniques including single-crystal X-ray analysis. Electronic absorption and fluorescence titration studies of R with different metal cations in a CH3CN/0.02 M HEPES buffer medium (pH = 7.3) show a highly selective binding affinity only toward Cu2+and Zn2+ ions even in the presence of other commonly coexisting ions such as Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cd2+, and Hg2+. Quantification of the fluorescence titration analysis shows that the chemosensor R can indicate the presence of Cu2+and Zn2+ even at very low concentrations of 17.3 and 16.5 ppb, respectively. R-Zn2+ acts as a selective metal-based fluorescent sensor for inorganic pyrophosphate ion (PPi) even in the presence of other common anions such as F-, Cl-, Br-, I-, CH3COO-, CO32-, HCO3-, N3-, SO42-, PPi, AMP, ADP, and ATP in an aqueous medium. The propensity of R as a bioimaging fluorescent probe to detect Cu2+ and Zn2+ ions in human cervical HeLa cancer cell lines and their cytotoxicity against human cervical (HeLa), breast cancer (MCF7), and noncancer breast epithelial (MCF10a) cells have also been investigated. R-Cu2+ shows better cytotoxicity and sensitivity toward cancer cells over noncancer cells than R and R-Zn2+ under identical conditions, with the appearance of apoptotic bodies. [ABSTRACT FROM AUTHOR]

The use of quantum dots (QDots) asbright and photostable probesfor long-term fluorescence imaging is gaining more interest. Thusfar, (pre)clinical use of QDots remains limited, which is primarilycaused by the potential toxicity of QDots. Most QDots consist of Cd2+ions, which are known to cause high levels of toxicity.In order to overcome this problem, several strategies have been tested,such as the generation of cadmium-free QDots. In the present study,two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect totheir cytotoxicity and cellular uptake in a variety of cell types.A multiparametric cytotoxicity approach is used, where the QDots arestudied with respect to cell viability, oxidative stress, cell morphology,stem cell differentiation, and neurite outgrowth. The data revealslight differences in uptake levels for both types of QDots (maximalfor QDotZnSe), but clear differences in cytotoxicity andcell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both typesof QDots can be explained by the intrinsic sensitivity of certaincell types and chemical composition of the QDots. At concentrationsat which no toxic effects can be observed, the functionality of theQDots for fluorescence cell visualization is evaluated, revealingthat the higher brightness of QDotZnSeovercomes most ofthe toxicity issues compared to that of QDotInP. Comparingthe results obtained with common Cd2+-containing QDotstested under identical conditions, the importance of particle functionalityis demonstrated, revealing that cadmium-free QDots tested in thisstudy are not significantly better than Cd2+-containingQDots for long-term cell imaging and that more work needs to be performedin optimizing the brightness and surface chemistry of cadmium-freeQDots for them to replace currently used Cd2+-containingQDots. [ABSTRACT FROM AUTHOR]

Although a lot of mitochondria-targeting biothiol probes have been developed and applied to cellular imaging through thiol-induced disulfide cleavage or Michael addition reactions, relatively few probes assess mitochondrial GSH with high selectivity over Cys and Hey and with NIR fluorescence capable of noninvasive imaging in biological samples. In order to monitor mitochondrial GSH with low background autofluorescence, we designed a heptamethine - azo conjugate as an NIR fluorescent probe by introducing a tunable lipophilic cation unit as the biomarker for mitochondria and a nitroazo group as the GSH-selective reaction site as well as the fluorescence quencher. The probe exhibited a dramatic off-on NIR fluorescence response toward GSH with high selectivity over other amino acids including Cys and Hey. Further application to cellular imaging indicated that the probe was highly responsive to the changes of mitochondrial GSH in cells. [ABSTRACT FROM AUTHOR]

In this study, we describe a solutionprocedure for the preparationand surface modification of photostable colloidal silicon quantumdots (SiQDs) for imaging of cancer cells. Photoluminescent SiQDs weresynthesized by reduction of halogenated silane precursors using amicroemulsion process. It was shown that 1,8-nonadiyne molecules couldbe grafted onto the surface of hydrogen-terminated SiQDs via ultraviolet(UV)-promoted hydrosilylation, demonstrated by Fourier transform infraredspectroscopy (FTIR) measurements. In addition, various azide moleculeswere coupled onto nonadiyne-functionalized particles, rendering particlesdispersible in selected polar and nonpolar solvents. The photoluminescenceof functionalized SiQDs was stable against photobleaching and didnot vary appreciably within biologically applicable pH and temperatureranges. To demonstrate compatibility with biological systems, water-solubleSiQDs were used for fluorescent imaging of HeLa cells. In addition,the SiQDs were shown to be non-cytotoxic at concentrations up to 240μg/mL. The results presented herein provide good evidence forthe versatility of functionalized SiQDs for fluorescent bioimagingapplication. [ABSTRACT FROM AUTHOR]

A range of biologically compatible, fluorescent rhenium-naphthalimide conjugates, based upon the rhenium factricarbonyl core, has been synthesized. The fluorescent ligands are based upon a N-functionalized, 4-amino-derived 1,8-naphthalimide core and incorporate a dipicolyl amine binding unit to chelate Re(I); the structural variations accord to the nature of the alkylated imide with ethyl ester glycine (L¹), 3-propanol (L²), diethylene glycol (L³), and benzyl alcohol (L4) variants. The species are fluorescent in the visible region between 505 and 537 nm through a naphthalimide-localized intramolecular charge transfer, with corresponding fluorescent lifetimes of up to 9.8 ns. The ligands and complexes were investigated for their potential as imaging agents for human osteoarthritic cells and protistan fish parasite Spironucleus vortens using confocal fluorescence microscopy. The results show that the specific nature of the naphthalimide structure serves to control the uptake and intracellular localization of these imaging agents. Significant differences were noted between the free ligands and complexes, with the Re(I) complex of L² showing hydrogenosomal localization in S. vortens. [ABSTRACT FROM AUTHOR]