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<p>Single-molecule localization microscopy enables high-resolution biological imaging, but its precision is limited by the rapid photobleaching of conventional fluorophores. Multicolour imaging is further constrained by the need for spectrally distinct dyes requiring separate excitations or sequential acquisition. Here we show that small (~10 nm) upconverting nanoparticles can be compositionally tuned to exhibit spontaneous, sustained blinking under single near-infrared excitation without optical or chemical modulation. By adjusting sensitizer (Yb<sup>3+</sup>)–emitter (Tm<sup>3+</sup>/Er<sup>3+</sup>) ratios, we identify a regime with intrinsic ON–OFF switching and low duty cycles (~0.9%) without photobleaching or statistical aging, enabling repeated localizations and sub-ångström precision (0.62 Å over 88,000 localizations) in upconversion-enabled stochastic optical reconstruction microscopy. By elucidating the underlying physical mechanism of this blinking, we engineered blue- and red-emitting probes for multicolour upconversion-enabled stochastic optical reconstruction microscopy. This technique enables the resolution of tightly packed UCNPs and the visualization of epidermal growth factor receptor dimers and multimers on cell membranes at single-protein resolution, all achieved with a simple optical setup without imaging buffers.</p>

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Spontaneous and indefinite blinking in upconverting nanoparticles for ångström-precision multicolour super-resolution imaging

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月27日

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  • Saptarshi Mandal,
  • Harrison W. Toll,
  • Kaibo Ma,
  • Irmuun Tommy Altankhuyag,
  • Lidao Li,
  • Wanlin Zhang,
  • João F. Shida,
  • Chunte Sam Peng

摘要

Single-molecule localization microscopy enables high-resolution biological imaging, but its precision is limited by the rapid photobleaching of conventional fluorophores. Multicolour imaging is further constrained by the need for spectrally distinct dyes requiring separate excitations or sequential acquisition. Here we show that small (~10 nm) upconverting nanoparticles can be compositionally tuned to exhibit spontaneous, sustained blinking under single near-infrared excitation without optical or chemical modulation. By adjusting sensitizer (Yb3+)–emitter (Tm3+/Er3+) ratios, we identify a regime with intrinsic ON–OFF switching and low duty cycles (~0.9%) without photobleaching or statistical aging, enabling repeated localizations and sub-ångström precision (0.62 Å over 88,000 localizations) in upconversion-enabled stochastic optical reconstruction microscopy. By elucidating the underlying physical mechanism of this blinking, we engineered blue- and red-emitting probes for multicolour upconversion-enabled stochastic optical reconstruction microscopy. This technique enables the resolution of tightly packed UCNPs and the visualization of epidermal growth factor receptor dimers and multimers on cell membranes at single-protein resolution, all achieved with a simple optical setup without imaging buffers.