STED microscopy represents an advanced approach to fluorescence imaging, enabling high spatial resolution. In our study, we examined the effectiveness of STED microscopy using three dyes simultaneously: ATTO 490LS DPPE, LIVE ORANGE Mito, and SiR Actin, which we used to label different cellular structures (membrane, mitochondria and actin filaments) in live fibroblasts isolated from mouse lung cells. We investigated the resolution achievable with confocal imaging and varying STED laser powers. With a STED laser power of 98 mW (30 %), we achieved a resolution below 100 nm for all three dyes, with the best resolution of approximately 76 nm using the LIVE ORANGE Mito dye. In the experiments, we observed issues with spectral crosstalk, where the signal from one dye leaked into the spectral windows of the other dyes. To address this issue, we applied a spectral decomposition methodology, which allowed effective separation of the dye signals, a key goal of our study. The signal of a dye in an incorrect spectral window was mainly reduced from 20 % to less than 5 %. Spectral decomposition enables the separation of fluorescent signals from complex images, aiding in the precise analysis of dye distribution and improving the visualization and interpretation of cellular structures. This technique can be used in studies of cellular dynamics, the identification of pathological changes, the evaluation of treatment efficacy, and the study of therapeutic responses. Due to its precision, it is an important technique in biophysics for understanding biological processes and structures.
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