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Area of Science:

  • Cell biology
  • Biophysics
  • Microscopy

Background:

  • Single-molecule detection in biological systems has advanced, but precise delivery of molecules to specific cellular locations remains a significant challenge.
  • Existing methods lack the ability to accurately position molecules and simultaneously observe cellular responses at the single-molecule level.

Purpose of the Study:

  • To develop and validate a novel imaging system for precise, localized delivery of single molecules to live cells.
  • To enable high-speed, 3D imaging of cellular responses to targeted molecular stimuli at single-molecule resolution.

Main Methods:

  • Combined light-sheet microscopy with nanopipette-based local delivery, termed local-delivery selective-plane illumination microscopy (ldSPIM).
  • Utilized nanopipette ionic feedback current for positional control and applied voltage for controlled molecule delivery.
  • Implemented single-objective SPIM with a reflective atomic force microscopy cantilever for precise single-molecule detection.

Main Results:

  • Demonstrated successful delivery of single fluorescently labeled proteins to the plasma membrane and cytoplasm of HK293 cells.
  • Showcased the system's capability by delivering amyloid-β aggregates to macrophages, enabling live imaging of MyDD88 accumulation and myddosome complex formation.
  • Observed aggregate-induced triggering of toll-like receptor 4 signaling in real-time.

Conclusions:

  • ldSPIM is a powerful, multifunctional imaging system for precise single-protein delivery and high-speed 3D imaging within live cells.
  • This technology overcomes previous limitations in localized molecular delivery, opening new avenues for studying cellular mechanisms.
  • The system facilitates detailed investigation of molecular interactions and signaling pathways at the single-molecule level.