Delivery of Fluorescent Nanoprobes into the Cytoplasm Using Cell-Penetrating Peptides for Live-Cell Super-Resolution Fluorescence Imaging

Vesicle entrapment remains a major bottleneck for the application of fluorescent nanoprobes (FNPs) with excellent optical properties in the field of super-resolution imaging of subcellular structures in living cells. Here, we developed a cell-penetrating peptide, fR8, which enables efficient cytoplasmic delivery of 3.5 nm FNPs instead of being trapped in vesicles, with a delivery efficiency of up to 75.6%. Further, we have successfully combined fusion protein technology with FNPs-fR8 to achieve high specificity and stable labeling of FNPs to various subcellular structures within live cells. Utilizing the advantages of FNPs, such as high brightness and resistance to photobleaching, FNPs modified with fR8 and the HaloTag ligand can be used for long-term super-resolution imaging of subcellular structures in living cells, allowing us to study the dynamic interactions between them. This work provides a practical tool for applying FNPs to live-cell super-resolution imaging and expands the application of super-resolution imaging technology in live-cell studies.