We designed and fabricated a light-powered nanoconverter for cytotoxicity toward human breast cancer cells. The nanoconverter was made from highly-fluorescent Ndoped carbon nanodots (C-dots), which were covalently conjugated to semiconductive hematite quantum dots (Qdots). The function of the nanoconverter was to transform the absorbed near-infrared (NIR) irradiation into reactive oxygen species (ROS) which would induce cell death. The principle of operation was based on the photosensitizing properties of Cdots, which have a two-photon absorption cross section. They absorbed NIR irradiation with wavelength in the range of 700−800 nm. The adsorbed energy was upconverted to photoluminescence that is emitted as higher-energy visible light with a maximum wavelength of ∼470 nm and transferred to the Q-dot moiety. The process was accompanied by ejection of electrons from the conduction band of Q-dots and by this mechanism, the nanoconverter produced aqueous superoxide anions, which oxidized organics and generated additional ROS. Our nanoconverter exposed in vitro to cultured human breast cancer HCC1954 cells induced light-dependent cell death as measured using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The cell death was minimal when the cells were exposed with C-dots alone or if the nanoconverter was exposed with the cells in dark.
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.
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