Fluorescent particle tracking is a powerful technique for studying intracellular transport and microrheological properties within living cells, which in most cases employs exogenous fluorescent tracer particles delivered into cells or fluorescent staining of cell organelles. Herein, we propose an alternative strategy, which is based on the generation of fluorescent species in situ with ultrashort laser pulses. Using mouse germinal vesicle oocytes as a model object, we demonstrate that femtosecond laser irradiation produces compact dense areas in the intracellular material containing fluorescent carbon dots synthesized from biological molecules. These dots have tunable persistent and excitation-dependent emission, which is highly advantageous for fluorescent imaging. We further show that tight focusing and tuning of irradiation parameters allow precise control of the location and size of fluorescently labeled areas and minimization of damage inflicted to cells. Pieces of the intracellular material down to the submicrometer size can be labeled with laser-produced fluorescent dots in real time and then employed as probes for detecting intracellular motion activity via fluorescent tracking. Analyzing their diffusion in the oocyte cytoplasm, we arrive to realistic characteristics of active forces generated within the cell and frequency-dependent shear modulus of the cytoplasm. We also quantitatively characterize the level of metabolic activity and density of the cytoskeleton meshwork. Our findings establish a new technique for probing intracellular mechanical properties and also promise applications in tracking individual cells in population or studies of spatiotemporal cell organization.

We propose a technique of controlled manipulation with mammalian intracellular bodies by means of optical trapping in order to reveal viscoelastic properties of cell interior. Near infrared laser in the spectral range of tissue transparency was applied to study dynamics of the nucleolus-chromatin complex inside the thermodynamically non-equilibrium system of a mouse oocyte. A nucleolus of germinal vesicle (GV) oocyte as spherical probe was displaced from the equilibrium and its relaxation dynamics was observed. We developed software for subdiffraction tracking of a nucleolus position with lateral resolution up to 3 nm and applied it for different GV-oocyte chromatin configurations. We showed differences in viscoelastic properties within nucleoplasm of NSN-oocytes, visualized by Hoechst 33342 staining. Also, we demonstrate that in germ cells basic biophysical properties of nucleoplasm can be obtained by using optical trapping without disruption and modification of cellular interior.

Laser synthesis of fluorescent species from biomolecules in living cells and tissues offers unique capabilities for fluorescent bioimaging, yet little is known about its mechanisms and characteristics of products. We examine synthesis of fluorescent products from water solution of L-lysine upon irradiation by trains of femtosecond laser pulses with varying parameters. We demonstrate that irradiation products contain nanoscale carbon-based fluorescent particles (carbon dots) with multi-colour and excitation-dependent emission. Morphology, chemical composition and fluorescent characteristics of irradiation products strongly depend on laser pulses parameters.

Owning to excellent optical properties and high bio-compatibility carbon dots (CDs) have drawn increasing attention and have been widely applied as imaging agents for various bio-applications. Here we report a strategy for live-cell fluorescent bioimaging based on in situ synthesis of CDs within cells by tightly focused femtosecond laser pulses. Laser-produced carbon dots exhibit bright excitationdependent fluorescence and are highly two-photon active under near infrared femtosecond excitation, thus demonstrating a potential for two-photon fluorescence imaging. The Raman spectra of fluorescent centers show strong D (1350 cm -1 ) and G (1590 cm -1 ) bands, thus suggesting that they are composed of carbon dots with sp 2 -hybridyzed core. Using Mouse GV oocytes as a model system we examine cytotoxicity and demonstrate the possibility of long-term fluorescent intracellular tracking of the laser-produced CDs. Created virtually in any point of the live cell, CD-based fluorescent µm-sized markers demonstrate high structural stability and retain bright fluorescence many hours after formation. Our results point to laser-produced fluorescent CDs as a highly-potent tool for cell cycle tracking, culture cell marking and probing intracellular movements.

Paracrine functions of mesenchymal stem (stromal) cells (MSCs) rely, at least partly, on membrane-bound extracellular vesicles (EVs) with rich composition of lipids, nucleic acids and signaling proteins. Elucidation the underlying chemistry could potentially lead to MSCs-free therapy. However, the secretome of MSCs (EVs’ composition) is non-static and depends on many other factors including surrounding cells and medium. Thus, the research techniques must be able to provide not only bulk but microscopy-scale data within a reasonable time frame. Two of these label-free techniques are subject of this work toward the question of chemical composition of the EVs.