Regioisomer-controlled red-shifted DNA probes for imaging of living tissues

Fluorescence imaging of chromatin DNA with high resolution and specificity is key to understanding cellular processes and enabling molecular diagnostics. However, choosing the best DNA probe for in vivo imaging is often a challenging task, as systematic studies investigating the biocompatibility of these molecules are lacking. Red-shifted fluorescent probes are particularly advantageous for imaging thick tissues, as light scattering decreases at red and far-red wavelengths. We synthesized and characterized the 4', 5', and 6' carboxyrhodamine regioisomers of SiR-Hoechst and 610CP-Hoechst DNA probes and assessed their performance in mice following intravenous injection. All variants entered tissues and produced stable staining that persisted for several hours ex vivo. In particular, 5' regioisomers show the highest performance, yielding bright and specific nuclear labeling. Renal clearance of the probes was nearly complete within 24 h, as indicated by fluorescence analysis of urine samples. Owing to their low cytotoxicity, high specificity, and favourable photophysical properties, these probes enabled high-quality confocal, two-photon, and STED microscopy in liver, kidney, lung, and heart tissue. Our findings extend the application of rhodamine-based DNA probes to in vivo imaging highlight their potential for deep-tissue imaging in live animals.