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An Optimized Protocol for SBEM-Based Ultrastructural Analysis of Cultured Human Cells
Natalia Diak1, Łukasz Chajec2, Agnieszka Fus-Kujawa1
1Department of Molecular Biology, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Medykow 18 Street, 40-752 Katowice, Poland.
Methods and Protocols
|August 27, 2025
Summary
We optimized serial block-face scanning electron microscopy (SBEM) for human cells. This new protocol enhances ultrastructural analysis of stem cells and fibroblasts, offering improved clarity and safety.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Biomedical Research
Background:
- Serial block-face scanning electron microscopy (SBEM) is crucial for 3D ultrastructural analysis.
- Its use with in vitro human cells is currently limited.
- Optimizing protocols is essential for broader application.
Purpose of the Study:
- To develop and validate an optimized SBEM sample preparation protocol for human cells.
- To improve the preservation and clarity of cellular ultrastructure.
- To establish a safe and efficient method for stem cells and fibroblasts.
Main Methods:
- Modified SBEM protocol using glutaraldehyde fixation and phosphate buffer.
- Application to human dermal fibroblasts and induced pluripotent stem cells (iPSCs).
- Validation using transmission electron microscopy (TEM).
Main Results:
- Excellent preservation of cellular ultrastructure with high contrast and clarity.
- Identified fixation during cell passage, not medium, as cause of morphology loss.
- Protocol demonstrated efficiency and safety.
Conclusions:
- The optimized SBEM protocol effectively analyzes human cell ultrastructure.
- This method is time-efficient, safe, and applicable to various human cell types.
- It provides a valuable tool for diverse biomedical research.
