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<p>The 3D spatiotemporal dynamics of tubular membrane protrusions are crucial for understanding phagocytosis, cellular communication and mechanobiology. Confocal microscopy, despite its prevalent use in membrane protrusion studies, presents limitations due to its inherently low axial resolution and high phototoxicity, which significantly hinder live imaging of tubular protrusion along the axial plane. We discovered that rotational oblique interference scattering (RO-iSCAT) leverages off-axis illumination to induce a larger lateral shift in out-of-focus iSCAT signals compared to in-focus signals. This phenomenon generates speckle-free widefield interferometric scattering signals with a 10-fold signal-to-noise ratio improvement, eliminating the need for any background subtraction. RO-iSCAT enables real-time, label-free imaging of diverse nanoparticles and tubular membrane protrusions, thus providing biophysical profiling of tubular membrane protrusions across multiple cell types and in complex co-cultures. RO-iSCAT empowers rapid quantitative dissection of the axial spatiotemporal complexities of membrane protrusions at tens to hundreds of nanometer displacements without requiring 3D volumetric imaging.</p>

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Using rotational integration of oblique interferometric scattering to track axial spatiotemporal responses of tubular membrane protrusions

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月14日

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  • Junyu Liu,
  • Yean Jin Lim,
  • David Herrmann,
  • Paul Timpson,
  • Tri Giang Phan,
  • Viviane Delghingaro-Augusto,
  • Christopher Richard Parish,
  • Huafeng Liu,
  • Min Guo,
  • Woei Ming Lee

摘要

The 3D spatiotemporal dynamics of tubular membrane protrusions are crucial for understanding phagocytosis, cellular communication and mechanobiology. Confocal microscopy, despite its prevalent use in membrane protrusion studies, presents limitations due to its inherently low axial resolution and high phototoxicity, which significantly hinder live imaging of tubular protrusion along the axial plane. We discovered that rotational oblique interference scattering (RO-iSCAT) leverages off-axis illumination to induce a larger lateral shift in out-of-focus iSCAT signals compared to in-focus signals. This phenomenon generates speckle-free widefield interferometric scattering signals with a 10-fold signal-to-noise ratio improvement, eliminating the need for any background subtraction. RO-iSCAT enables real-time, label-free imaging of diverse nanoparticles and tubular membrane protrusions, thus providing biophysical profiling of tubular membrane protrusions across multiple cell types and in complex co-cultures. RO-iSCAT empowers rapid quantitative dissection of the axial spatiotemporal complexities of membrane protrusions at tens to hundreds of nanometer displacements without requiring 3D volumetric imaging.