Elucidating the dynamics of DNA repair proteins is essential to understanding the mechanisms that preserve genomic stability and prevent carcinogenesis. However, the measurement and modeling of protein dynamics at DNA lesions via currently available image analysis tools is cumbersome. Therefore, we developed CellTool—a stand-alone open-source software with a graphical user interface for the analysis of time-lapse microscopy images. It combines data management, image processing, mathematical modeling, and graphical presentation of data in a single package. Multiple image filters, segmentation, and particle tracking algorithms, combined with direct visualization of the obtained results, make CellTool an ideal application for the comprehensive analysis of DNA repair protein dynamics. This software enables the fitting of obtained kinetic data to predefined or custom mathematical models. Importantly, CellTool provides a platform for easy implementation of custom image analysis packages written in a variety of programing languages. Using CellTool, we demonstrate that the ALKB homolog 2 (ALKBH2) demethylase is excluded from DNA damage sites despite recruitment of its putative interaction partner proliferating cell nuclear antigen (PCNA). Further, CellTool facilitates the straightforward fluorescence recovery after photobleaching (FRAP) analysis of BRCA1 associated RING domain 1 (BARD1) exchange at complex DNA lesions. In summary, the software presented herein enables the time-efficient analysis of a wide range of time-lapse microscopy experiments through a user-friendly interface.

The development of fluorescence-based methods for bioassays and medical diagnostics requires the design and synthesis of specific markers to target biological microobjects. However, biomolecular recognition in real cellular systems is not always as selective as desired. A new concept for creating fluorescent biomolecular probes, utilizing a fluorogenic dye and biodegradable, biocompatible nanomaterials, is demonstrated. The synthesis of a new dicationic asymmetric monomethine cyanine dye with benzo[d]thiazolium-N-propionamide and chloroquinoline end groups is presented. The photophysical properties of the newly synthesized dye were examined through the combined application of spectroscopic and theoretical methods. The applicability of the dye as a fluorogenic nucleic acid probe was proven by UV-VIS spectroscopy and fluorescence titration. The dye–nucleic acid interaction mode was investigated by UV-Vis and CD spectroscopy. The newly synthesized dicationic dye, like other similar fluorogenic structures, limited permeability, which restricts its use as a probe for RNA and DNA. To enhance cellular delivery, we utilized a patented technology that employs solid, insoluble lipid nanoparticles. This method ensures the complete introduction of the dye into cells while minimizing activity outside the cells. In our study involving two human cell lines, we observed improved penetration through the cell membrane and distinctive selectivity in visualizing nucleic acids within the cytoplasm and nucleus.