Radmacher Niels, Chizhik Alexey I, Nevskyi Oleksii, Gallea José Ignacio, Gregor Ingo, Enderlein Jörg
Third Institute of Physics - Biophysics, Georg August University of Göttingen, Göttingen, Germany; email:
Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), Universitätsmedizin Göttingen, Göttingen, Germany.
Annu Rev Biophys. 2025 May;54(1):163-184. doi: 10.1146/annurev-biophys-071524-105321. Epub 2025 Feb 14.
Over the last 30 years, fluorescence microscopy, renowned for its sensitivity and specificity, has undergone a revolution in resolving ever-smaller details. This advancement began with stimulated emission depletion (STED) microscopy and progressed with techniques such as photoactivatable localization microscopy and stochastic optical reconstruction microscopy (STORM). Single-molecule localization microscopy (SMLM), which encompasses methods like direct STORM, has significantly enhanced image resolution. Even though its speed is slower than that of STED, SMLM achieves higher resolution by overcoming photobleaching limitations, particularly through DNA point accumulation for imaging in nanoscale topography (DNA-PAINT), which continuously renews fluorescent labels. Additionally, cryo-fluorescence microscopy and advanced techniques like minimal photon fluxes imaging (MINFLUX) have pushed the boundaries toward molecular resolution SMLM. This review discusses the latest developments in SMLM, highlighting methods like resolution enhancement by sequential imaging (RESI) and PAINT-MINFLUX and exploring axial localization techniques such as supercritical angle fluorescence and metal-induced energy transfer. These advancements promise to revolutionize fluorescence microscopy, providing resolution comparable to that of electron microscopy.
在过去30年里，以其灵敏度和特异性而闻名的荧光显微镜在解析越来越小的细节方面经历了一场革命。这一进展始于受激发射损耗（STED）显微镜，并随着光激活定位显微镜和随机光学重建显微镜（STORM）等技术的发展而不断推进。单分子定位显微镜（SMLM），包括直接STORM等方法，显著提高了图像分辨率。尽管其速度比STED慢，但SMLM通过克服光漂白限制实现了更高的分辨率，特别是通过用于纳米级形貌成像的DNA点积累（DNA-PAINT），它能不断更新荧光标记。此外，低温荧光显微镜和最小光子通量成像（MINFLUX）等先进技术已将界限推向分子分辨率的SMLM。本文综述讨论了SMLM的最新进展，重点介绍了通过顺序成像提高分辨率（RESI）和PAINT-MINFLUX等方法，并探索了超临界角荧光和金属诱导能量转移等轴向定位技术。这些进展有望彻底改变荧光显微镜，提供与电子显微镜相当的分辨率。
