Nature biotechnology · 2026
Mirror-enhanced 4Pi-SMLM with one objective enables isotropic nanoscale imaging.
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Yu Zijing, Zheng Bei, Zhan Yajing, Li Shuxin, Wang Xulong, Dai Qiuyang, Chen Yanqin, Wei Zongfang, Zhao Wenxuan, Chen Linlin, Tang Jiaxin, Xia Tianchang, He Lingjuan, Liu Changliang, Wu Xudong, Yu Xiaochun, Zhang Yongdeng
Аннотация
Dual-objective 4Pi single-molecule localization microscopy (4Pi-SMLM) offers isotropic nanoscale resolution; however, its broader adoption is limited by instrumental complexity and stringent alignment requirements. Here we introduce mirror-enhanced 4Pi-SMLM (me4Pi-SMLM), a single-objective configuration that uses mirror-based retroreflection of the illumination beam to generate phase-tunable interference fringes. This design improves the axial resolution of astigmatism-based methods by approximately fivefold, delivering performance comparable to conventional 4Pi-SMLM while greatly reducing system complexity and maintenance. me4Pi-SMLM achieves near-isotropic localization precision of 2-3 nm in biological samples, enabling clear and unambiguous visualization of diverse ultrastructural features. Furthermore, it achieves sub-15 nm isotropic resolution in brain slices and facilitates high-fidelity two-colour imaging, nanoscale whole-cell reconstruction and live-cell imaging. me4Pi-SMLM can be seamlessly integrated into existing 3D-SMLM systems, enhancing performance with minimal cost and effort.