biostudies-literatureZhang CFundamental Research Funds for the Central UniversitiesNational Natural Science Foundation of China10255-10261https://www.ebi.ac.uk/biostudies/studies/S-EPMC10975311biostudies-literatureUnknown14(15)Fluorescence imaging of organelles at the cellular level is important for studying biological processes. The development of a highly emissive fluorescent probe that operates under a suitable excitation light source is a key step in high-quality fluorescence imaging. For long-term, high-fidelity fluorescence imaging of mitochondria-related cellular processes using two-photon microscopy and stimulated emission depletion microscopy, we developed a new benzocoumarin-based cationic fluorescent probe (BS-CN) that is far-red emitting, water-soluble, photostable, and very bright in cells. BS-CN showed a remarkably high quantum yield of 0.35 and a large two-photon excited fluorescence action cross-section of 76 GM, enabling the long-term tracking of mitochondria in live cells. In addition, BS-CN exhibited a certain affinity for RNA and stained nucleoli in fixed cells. A comparative assessment of the photophysical properties and bioimaging performance of benzo[<i>h</i>]coumarin-pyridinium and the structurally similar styryl-pyridinium (BS-MN) clearly indicated the importance of structural rigidity for fluorescence efficiency.RSC advancesRigidify styryl-pyridinium dyes to benzo[<i>h</i>]coumarin-based bright two-photon fluorescent probes for cellular bioimaging.PMC10975311U23301062022JC00352350002Liu YJin WLiu ZZhang CYu ZYu XfalseRigidify styryl-pyridinium dyes to benzo[<i>h</i>]coumarin-based bright two-photon fluorescent probes for cellular bioimaging.Fluorescence imaging of organelles at the cellular level is important for studying biological processes. The development of a highly emissive fluorescent probe that operates under a suitable excitation light source is a key step in high-quality fluorescence imaging. For long-term, high-fidelity fluorescence imaging of mitochondria-related cellular processes using two-photon microscopy and stimulated emission depletion microscopy, we developed a new benzocoumarin-based cationic fluorescent probe (BS-CN) that is far-red emitting, water-soluble, photostable, and very bright in cells. BS-CN showed a remarkably high quantum yield of 0.35 and a large two-photon excited fluorescence action cross-section of 76 GM, enabling the long-term tracking of mitochondria in live cells. In addition, BS-CN exhibited a certain affinity for RNA and stained nucleoli in fixed cells. A comparative assessment of the photophysical properties and bioimaging performance of benzo[<i>h</i>]coumarin-pyridinium and the structurally similar styryl-pyridinium (BS-MN) clearly indicated the importance of structural rigidity for fluorescence efficiency.2024-01-01T00:00:00Z2024 Mar2025-04-26T11:24:07.571Z2025-04-06T13:42:00.933ZS-EPMC109753113854979410.1039/d3ra08269e