Cellular and Intravital Nucleus Imaging by a D-π-A Type of Red-Emitting Two-Photon Fluorescent Probe

The advancement in fluorescent probe technology for visualizing nuclear morphology and nucleic acid distribution in live cells and in vivo has attracted considerable interest within the biomedical research community, as it offers invaluable insights into cellular dynamics across various physiological and pathological contexts. In this study, we present a novel two-photon nucleus-imaging fluorescent probe called Nu-red, which is a typical donor(D)-π-acceptor(A) rotor composed of the donor (dihydroquinoline) and acceptor (pyridiniumylpentadienitrile) parts linked by a single bond. This probe offers several advantages, including long-wavelength excitation and emission (λ_ex/λ_em = 610/664 nm), favorable quantum yields (1.35-22.15%), excellent two-photon absorption cross-section (425.92 GM), high selectivity and sensitivity, high DNA-binding affinity (K_a = 3.7 × 10⁷ M^-1, comparable to that of the commercial nucleus stain Hoechst 33342), rapid entry into the nucleus (1 min), low cytotoxicity, membrane-permeability, good water solubility, applicability to various cell lines, and compatibility with other commercial probes. Leveraging these aforementioned advantages, Nu-red was successfully employed to visualize cell division in living cells, distinguish abnormal division cells from normal ones, and track morphological changes in the nucleus during cell apoptosis. More notably, Nu-red was utilized to visualize nuclear shrinkage and pyknosis in the brain of a living mouse model of ischemic stroke.