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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
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.
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.
