Ervin Kovács

Novel cryptocyanine‐based DNA‐binding fluorescent probes were developed by introducing side chains with varying polarity to the dye scaffold. This structural modification improves water solubility, reduces aggregation in aqueous media, and enhances DNA binding affinity. Upon binding to DNA, the derivatives exhibit a high increase in fluorescence quantum yield, demonstrating their potential as fluorogenic DNA probes. The photophysical behavior of the dyes is systematically investigated using spectroscopic techniques, focusing on their environment‐sensitive emission properties. These results highlight the importance of environmentally responsive dye scaffolds in the development of fluorogenic tools for nucleic acid detection and diagnostic applications.

Novel pyrylium-based fluorescent DNA-binding dyes were developed, containing p-aminoaryl groups at the α positions of the pyrylium moiety. The amines are substituted with aminoalkyl groups to enhance the dye's water solubility and DNA binding affinity. With this structural modification triarylpyryliums exhibit up to 7.6 times higher fluorescence enhancement upon binding to DNA, rendering them promising candidates for fluorogenic DNA sensors. The photochemical characteristics of the dyes, including the radiationless deexcitation pathways necessary for the DNA-induced fluorogenicity were studied using both spectroscopical experiments and theoretical calculations. Particular attention was given to the possibility of twisted intramolecular charge transfer (TICT) states as a quenching pathway in the unbound state. These findings support the rational design of structurally dynamic, environment-sensitive fluorophores for nucleic acid detection and molecular diagnostics.

A new family of azaborine fluorophores, consisting of ten compounds, has been developed that mimics the chromophore of green fluorescent protein, with conformation locked by a hydroxyboron bridge. These fluorophores exhibit fluorescence Stokes shifts up to 100 nm (0.56 eV) and high brightness (>10⁴ M^-1 cm^-1) in the 408-525 nm range. Their potential utility for sensing applications was demonstrated by constructing zinc sensors capable of detecting 100 micromolar Zn²⁺ concentrations in biological systems with two-photon microscopy.

The advancements in targeted drug release and experimental neuroscience have amplified the scientific interest in photolabile protecting groups (PPGs) and photouncaging. The growing need for the detection of uncaging events has led to the development of reporters with fluorescence turn-on upon uncaging. In contrast, fluorescent tags with turn-off properties have been drastically underexplored, although there are applications where they would be sought after. In this work, a rhodamine-based fluorescent tag is developed with signal turn-off following photouncaging. One-photon photolysis experiments reveal a ready loss of red fluorescence signal upon UV (365 nm) irradiation, while no significant change is observed in control experiments in the absence of PPG or with irradiation around the absorption maximum of the fluorophore (595 nm). The two-photon photolysis of the turn-off fluorescent tag is explored in hydrogel photolithography experiments. The hydrogel-bound tag enables the power-, dwell time-, and wavelength-dependent construction of intricate patterns and gradients. Finally, a prominent caged neurotransmitter (MNI-Glu) is modified with the fluorescent tag, resulting in the glutamate precursor named as GlutaTrace with fluorescence traceability and turn-off upon photouncaging. GlutaTrace is successfully applied for the visualization of glutamate precursor distribution following capillary microinjection and for the selective excitation of neurons in a mouse brain model.

Background: The organism-wide effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. It has been reported that SARS-CoV-2 infection pathways converge at calcium influx and subcellular calcium distribution changes. Imaging combined with a proper staining technique is an effective tool for studying subcellular calcium-related infection and replication mechanisms at such resolutions. Methods: Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells. Results: The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load. Conclusions: We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies. Graphical