1. Cell-permeable organic fluorescent probes for live-cell super-resolution imaging

Recent developments in single-molecule localization microscopy (SMLM), such as photo-activated localization microscopy and stochastic optical reconstruction microscopy, have enabled biological structures to be defined with a spatial resolution beyond the diffraction limit, providing powerful techniques to obtain exciting new insight into the nanoscale structures and dynamics of biological samples. SMLM does not require sophisticated microscopes but instead critically relies on the use of photoactivatable, photo-convertible or photo-switchable (termed photo-modulatable) fluorescent probes.

Professor Zhang’s research team have included the cell-penetrating peptides into the construction of photo-modulatable fluorescent probes for the first time and finally developed a series of new cell-permeable photo-modulatable fluorescent probes, which can directly label intracellular endogenous targeted proteins in live cells for super-resolution imaging. By using the TIRF super-resolution imaging system and the PALMER algorithm for high density localization, which developed by Professor Huang’s research team, they have obtained super-resolution images of F-actin and lysosomes in live cells and for the first time recorded the dynamics process of a connection rearrangement of F-actin under physiological conditions. The probes in the research are the first cell-permeable photo-modulatable organic fluorescent probes that can directly label intracellular endogenous targeted proteins in live cells for super-resolution imaging. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.

2. Application: live-cell long-term super-resolution imaging reveals lysosome-mitochondrion interactions

Increasing evidence indicates that intracellular organelles are integrated into cellular networks and cooperate in diverse cellular tasks rather than acting as isolated entities. Specialized membrane contact sites, usually on a nanometer scale, are formed between organelles, providing distinct spatial regions for executing and regulating their functions. However, only limited studies of the dynamic physical interactions between organelles have been reported, leading to an incomplete understanding of the interplay between organelles. The recent development of structured illumination microcopy (SIM), which offers substantially improved spatiotemporal resolution, provides a powerful tool for characterizing the dynamics of subcellular structures in live cells. However, limitations of the existing fluorescent probes for organelles, such as cell-impermeable, photobleaching, and a nonspecific background, hinder the characterization of dynamic physical interactions between organelles in live cells.

Here, we develop a series of novel cell-permeable organic fluorescent probes for organelles with high specificity and excellent photostability. Using our probe, we captured the dynamic processes of lysosomal fission and fusion in live cells by super-resolution imaging for the first time. Our results directly visualized the entire process of lysosomal fusion and the subsequent re-formation of lysosomes, therefore providing new insights into the dynamic behavior of lysosomes in vivo. We also successfully recorded the dynamic process of physical lysosome-mitochondrion interactions in live cells at 6-s intervals with a spatial resolution of approximately 90 nm. We identified four types of dynamic physical interactions between lysosomes and mitochondria. Among these interactions, we characterized an entire dynamic process (as long as 13 min) of mitochondrial transfer between two lysosomes, providing visual evidence of dynamic physical lysosome-mitochondrion interactions at a high spatiotemporal resolution and over a long time course. To the best of our knowledge, this is the first time that dynamic physical interactions between lysosomes and mitochondria have been characterized and that the existence of contact sites between them has been revealed in live cells. Our probes provide an avenue for understanding the functions and the dynamic interplay of organelles in live cells.