Dekaliuk M. New nanocarbon fluorophores: synthesis, optical properties and their interactions with cells.

The thesis is devoted to research of the new type of fluorescent carbon nanomaterials - carbon dots (CNDs). We have demonstrated different approaches to their synthesis and have studied the structural and optical features. Moreover, potential ways for their application in fluorescence microscopy were proposed. The carbon nanoparticles were prepared from alanine, citric acid, urea, etc. by hydrothermal treatment. As the fluorescent materials, the obtained structures are brightly fluorescent in the visible region, soluble in various organic solvents, stable at different pH and are non-toxic for cell cultures. The CNDs demonstrate the presence of distribution of fluorescence properties of individual fluorophores that behave as individual emitters and do not exchange their excited-state energies via homo-FRET. The time-resolved studies demonstrate heterogeneity of fluorescence decays occurring in nanosecond time range. Besides, it was found that carbon dots represent the assemblies of individual emitters possessing variable fluorescent properties including optical anisotropy, limited rotational mobility and high accessibility to ionic quenchers. We present new results on a comprehensive photoluminescence study of single carbon dots nanoparticles with high resolution transmission electron microscopy and atomic force microscopy measurements. The power law character of CND blinking along with their multistate time traces suggests that the charge trapping and its redistribution on the surface of the particles triggers their transitions between emissive and dark states. Thus, CNDs possess a unique hybrid combination of fluorescence properties, exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The selective cell labeling with CNDs was demonstrated. With these tools we demonstrate that both native and apoptotic cells can be easily visualized. The CNDs uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. Using different methods of sample preparation, the ability for labeling various structural compartments of the cell was shown. For living cells there are the intracellular vesicles and lysosomes. In contrast, in fixed cells the nucleus is labeled preferentially. The fact that apoptotic cells accumulate strongly increased amount of CNDs can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. The application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. Moreover, our studies showed the possibility of using these nanoscale fluorophores for superresolution method SOFI. The technique is conceptually di?erent from localization based methods and has a number of advantages. The primary ones are relaxed blinking requirements and inherent background elimination. This allowed us to use the method without any additional modi?cation of the as-synthesized CNDs.This approach improves the detailization of fluorescent images by 30 percent.