Dynamic Monitoring of Organelle Interactions in Living Cells via Two-Color Digitally Enhanced Stimulated Emission Depletion Super-resolution Microscopy

One of the most significant advances in stimulated emission depletion (STED) super-resolution microscopy is its capacity for dynamic super-resolution imaging of living cells, including the long-term tracking of interactions between various cells or organelles. Consequently, the multicolor STED plays a pivotal role in biological research. Despite the emergence of numerous fluorescent probes characterized by low toxicity, high stability, high brightness, and exceptional specificity, enabling dynamic imaging of living cells with multicolor STED, practical implementation of multicolor STED for live-cell imaging is influenced by several factors. These factors include the power and wavelength of the STED beam, the duration of imaging, the size of the imaging area, and the complexity of sample preparation. Presently, a major limitation of multicolor STED is the requirement for high STED power, which hinders the monitoring of interactions between different cells or organelles due to the associated irreversible optical damage. To address this issue, this paper emphasizes research findings based on the digitally enhanced STED (DE-STED) technique. This method overcomes the aforementioned challenge by utilizing low STED laser power to achieve prolonged two-color STED super-resolution imaging of living cells, effectively mitigating phototoxic effects and enhancing the capacity to observe intracellular dynamics. With a depletion laser power of less than 1 mW, we achieved a resolution of about 87 nm, close to that achievable with conventional high-power STED technology.