Improving Brightness and Stability of Si-Rhodamine for Super-Resolution Imaging of Mitochondria in Living Cells

Photostable and bright organic dyes emitting in the near-infrared region are highly desirable for long-term dynamic bioimaging. Herein, we report a synthetic approach to build novel methoxy modified Si-rhodamine (SiRMO) dyes by introducing the methoxybenzene on the xanthene moiety. The brightness of SiRMO increased from 2300 M^–1 cm^–1 (SiRMO-0) to 49000 M^–1 cm^–1 (SiRMO-2) when the substituent 2,5-dimethoxybenzene was replaced with 2,6-dimethoxybenzene. Moreover, the stability of SiRMO-2 was significantly improved due to the steric hindrance protection of the two methoxy groups on the ninth carbon atom of the xanthene. After fast cellular uptake, the SiRMO dyes selectively stained the mitochondria with a low background in live cultured cells and primary neurons. The high brightness and stability of SiRMO-2 significantly improved the capability of monitoring mitochondria dynamic processes in living cells under super-resolution conditions. Moreover, with the fluorescence nanoscopy techniques, we observed the structure of mitochondrial cristae and mitochondria fission, fusion, and apoptosis with a high temporal resolution. Under two-photon illumination, SiRMO-2 showed also enhanced two-photon brightness and stability, which are important for imaging in thick tissue.