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<p>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.</p>

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Mirror-enhanced 4Pi-SMLM with one objective enables isotropic nanoscale imaging

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月31日

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  • Zijing Yu,
  • Bei Zheng,
  • Yajing Zhan,
  • Shuxin Li,
  • Xulong Wang,
  • Qiuyang Dai,
  • Yanqin Chen,
  • Zongfang Wei,
  • Wenxuan Zhao,
  • Linlin Chen,
  • Jiaxin Tang,
  • Tianchang Xia,
  • Lingjuan He,
  • Changliang Liu,
  • Xudong Wu,
  • Xiaochun Yu,
  • Yongdeng Zhang

摘要

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.