Related Experiment Video
Updated: Sep 26, 2025
14:19
2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development
Published on: March 25, 2022
4.1K
3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines
Kari-Anne M Frikstad1, Kay O Schink2, Sania Gilani1,2
1Department of Radiation Biology, Institute of Cancer Research, OUH-Norwegian Radium Hospital, Oslo, Norway.
Bio-Protocol
|April 18, 2022
Summary
This study presents a 3D-structured illumination microscopy protocol for high-resolution mapping of centrosomal proteins. The method precisely localizes proteins within the centrosome, aiding research into human diseases linked to centrosome and primary cilia dysfunction.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Human Genetics
Background:
- The centrosome, a key microtubule-organizing center, is crucial for cell division and primary cilium formation.
- Aberrations in centrosomes and cilia are implicated in human genetic disorders and cancer.
- Accurate mapping of centrosomal proteins is vital for understanding these diseases.
Purpose of the Study:
- To develop and validate a high-resolution 3D-structured illumination microscopy (3D-SIM) protocol for centrosomal protein localization.
- To enable comparative analysis of protein distribution within centrosome substructures.
- To facilitate the study of disease-associated centrosomal proteins and their mutations.
Main Methods:
- Detailed 3D-SIM protocol for fixed human cell lines.
- Optimization for antibodies requiring harsh fixation, preserving cellular architecture.
- Compatibility with fluorescent protein tags and implementation of an internal reference for 3D channel alignment.
Main Results:
- Achieved approximately 120 nm lateral and 300 nm axial resolution for centrosomal protein localization.
- Demonstrated preservation of centriole and centrosome architecture compared to TEM.
- Validated the protocol's utility for analyzing protein interdependence and mutations.
Conclusions:
- The developed 3D-SIM protocol offers a flexible, rapid, and informative method for centrosomal protein analysis.
- This technique enhances the study of centrosome function and its role in human health and disease.
- Enables precise mapping of disease-associated proteins and mutations within the centrosome.
