Shen Xiaochun, Wang Luwei, Guo Yong, Wang Chenguang, Yan Wei, Qu Junle
College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, State Key Laboratory of Radio Frequency Heterogeneous Integration (Shenzhen University), Shenzhen University, Shenzhen 518060, P. R. China.
State Key Laboratory of Integrated Optoelectronics, College of Electronic Science & Engineering, Jilin University, Changchun 130012, P. R. China.
J Phys Chem Lett. 2025 Jan 16;16(2):596-603. doi: 10.1021/acs.jpclett.4c03326. Epub 2025 Jan 7.
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.
受激发射损耗（STED）超分辨率显微镜最重要的进展之一是其对活细胞进行动态超分辨率成像的能力，包括对各种细胞或细胞器之间相互作用的长期跟踪。因此，多色STED在生物学研究中起着关键作用。尽管出现了许多具有低毒性、高稳定性、高亮度和卓越特异性的荧光探针，使得能够用多色STED对活细胞进行动态成像，但多色STED用于活细胞成像的实际应用受到几个因素的影响。这些因素包括STED光束的功率和波长、成像持续时间、成像区域的大小以及样品制备的复杂性。目前，多色STED的一个主要限制是需要高STED功率，这由于相关的不可逆光学损伤而阻碍了对不同细胞或细胞器之间相互作用的监测。为了解决这个问题，本文重点介绍基于数字增强STED（DE-STED）技术的研究成果。该方法通过利用低STED激光功率实现对活细胞的长时间双色STED超分辨率成像，有效减轻光毒性效应并增强观察细胞内动力学的能力，从而克服了上述挑战。在耗尽激光功率小于1 mW的情况下，我们实现了约87 nm的分辨率，接近传统高功率STED技术所能达到的分辨率。
