The real time monitoring and quantification of the concentration of highly fluorescent nitrogendoped carbon nanodots (C-dots) in eukaryotic Tobacco bright yellow-2 (BY-2) plant cells was investigated by fluorescence and confocal microscopy. The quantitative measurement of their fluorescent emission intensity was possible because of the high photo-resistance, good water solubility and the absence of fading effect of the nanoparticles, which is frequent occurred problem of the conventional organic dyes. The microscopic analysis revealed that C-dots entered generally into the cells through endocytosis and caused negligible cytotoxicity. The multicolor cellular imaging of labeled Tobacco BY-2 demonstrates that the cells were in good health conditions and any blinking artifacts were not observed. The quantification of fluorescence emission intensity was carried out in the intracellular regions where the relationship between the C-dots concentration and relative emission was linear. Based on a control experiment with fluorescence liposomes with known dependence between C-dots concentration and emission, we were able to determine the amount of accumulated nanoparticles in the inner compartments of the eukaryotic cell through subsequent digital image analysis. The reported microscopic approach may be used for accurate testing and direct examination of the drug internalization mechanisms by C-dots as sensitive probes in single cells or tissues. K E Y W O R D Scarbon nanodots, multicolor intracellular imaging, nanoparticles quantification
The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL-14-dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high-resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ∼ 10 mmol/kg in area of 0.1 μm , which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase-plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase-plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.
The object of the present report is to provide a method for a visualization of DNA in TEM by complementary labeling of cytosine with guanine derivative, which contains platinum as contrast-enhanced heavy element. The stretched single-chain DNA was obtained by modifying double-stranded DNA. The labeling method comprises the following steps: (i) stretching and adsorption of DNA on the support film of an electron microscope grid (the hydrophobic carbon film holding negative charged DNA); (ii) complementary labeling of the cytosine bases from the stretched single-stranded DNA pieces on the support film with platinum containing guanine derivative to form base-specific hydrogen bond; and (iii) producing a magnified image of the base-specific labeled DNA. Stretched single-stranded DNA on a support film is obtained by a rapid elongation of DNA pieces on the surface between air and aqueous buffer solution. The attached platinum-containing guanine derivative serves as a high-dense marker and it can be discriminated from the surrounding background of support carbon film and visualized by use of conventional TEM observation at 100 kV accelerated voltage. This method allows examination of specific nucleic macromolecules through atom-by-atom analysis and it is promising way toward future DNA-sequencing or molecular diagnostics of nucleic acids by electron microscopic observation.
The paper focuses on the development of ultra-small carbon nanodots as sensitive sensors for detection and monitoring of manganese in drinking water and groundwater. The environmental contamination with manganese is often a result from industrial activities such as mining and agriculture. The soluble Mn ions are toxic for many organisms and might even cause serious human diseases. The strategy for sensory detection used by us is based on a photo-oxidation reaction, in which the carbon nanodots are dissolved in acidic solution and illuminated with UV irradiation ( = 365 nm). At these conditions the nanoparticles acted as photosensitizer and produced singlet oxygen, which can be monitored by 3,3’,5,5’-tetramethylbenzidine as an organic indicator. However, to proceed this reaction, it is necessary to introduce the redox pair Mn(II)/Mn(III) in the reaction mixture, which plays the role of a catalytic mediator for the electron transfer. Thus, the oxidation of 3,3’,5,5’-tetramethylbenzidine appears, which results in blue colour of the solution with absorbance maximum at 645 nm. The rate of this sensory reaction was significantly enhanced in the presence of EDTA, which was used as a ligand to obtain a stable complex of Mn(III)/EDTA. The formed complex itself oxidised the organic indicator and caused the colorimetric reaction. Among the other metal ions tested, only manganese could be detected by the presented sensory approach.
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