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An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed

Yujia Qiu1, Elma Sakinatus Sajidah2, Sota Kondo1

  • 1Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan.

Cells
|February 9, 2024
PubMed
Summary

This study introduces a new method for observing nuclear pore complex dynamics in mouse brain cells using high-speed atomic force microscopy. This technique allows for nanoscale imaging of these crucial cellular structures in near-native states.

Keywords:
high-speed atomic force microscopy (HS-AFM)mouse brainnuclear porestrainer

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Area of Science:

  • Cell Biology
  • Biophysics
  • Nanotechnology

Background:

  • Nuclear pore complexes (NPCs) regulate transport between the nucleus and cytoplasm.
  • Existing imaging methods like optical microscopy lack nanoscale resolution, and super-resolution techniques often require fixed samples, limiting dynamic studies.
  • High-speed atomic force microscopy (HS-AFM) offers nanoscale imaging of molecules in near-native states but faces challenges in mammalian tissue preparation.

Purpose of the Study:

  • To develop a reliable method for nanoscale imaging of nuclear pore complex dynamics in mammalian cells.
  • To overcome sample preparation limitations for HS-AFM in brain tissue.
  • To visualize the spatiotemporal dynamics of nuclear pores in real-time.

Main Methods:

  • Development of the rapid strainer microfiltration (RSM) protocol for preparing high-quality mouse brain nuclei.
  • Utilization of HS-AFM for real-time imaging and cinematography.
  • Nanoscale observation of nuclear pore complexes in their native environment.

Main Results:

  • Successful preparation of high-quality mouse brain nuclei using the RSM protocol.
  • Real-time nanoscale imaging of nuclear pore dynamics achieved using HS-AFM.
  • Acquisition of spatiotemporal data on nuclear pore nano-dynamics.

Conclusions:

  • The RSM protocol combined with HS-AFM provides a powerful approach for studying nuclear pore dynamics at the nanoscale.
  • This method enables the observation of cellular processes in near-native conditions, overcoming limitations of traditional techniques.
  • The findings offer new insights into the dynamic nature of nuclear pore complexes in mammalian systems.