Characterizing the long-term nanometer-scale interactions between lysosomes and mitochondria in live cells is essential for understanding their functions but remains challenging due to limitations of the existing fluorescent probes. Here, we develop cell-permeable organic fluorescent probes for lysosomes with excellent specificity and high photostability. We also use an existing Atto 647N dye with high brightness and excellent photostability to achieve specific labeling of mitochondria in live cells. Using these probes, we obtain dual-color structured illumination microscopy (SIM) images of dynamic physical lysosome-mitochondrion interactions in live cells at an ~90-nm resolution over a long time course of ~13 min. We successfully record the consecutive dynamic processes of lysosomal fusion and fission, as well as four types of physical lysosome-mitochondrion interactions by super-resolution imaging. Our probes provide an avenue for understanding the functions and the dynamic interplay of lysosomes and mitochondria in live cells.

Imaging and tracking of near-surface three-dimensional volumetric nanoscale dynamic processes of live cells remains a challenging problem. In this paper, we propose a multi-color live-cell near-surface-volume super-resolution microscopy method that combines total internal reflection fluorescence structured illumination microscopy with multi-angle evanescent light illumination. We demonstrate that our approach of multi-angle interference microscopy is perfectly adapted to studying subcellular dynamics of mitochondria and microtubule architectures during cell migration.

Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)₃R₂. We develop photo-modulatable organic fluorescent probes consisting of a (rR)₃R₂ peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.