Uranium-Free KMnO₄/Pb Staining Enables Imaging of Tissue Architectures in Low Vacuum Scanning Electron Microscopy

Academic Background

Electron Microscopy (EM) is one of the most powerful tools for studying the ultrastructure of cells and tissues. However, traditional metal staining methods for biological samples often require the use of harmful uranium compounds, which limits the widespread application of electron microscopy. In recent years, with the development of super-resolution fluorescence microscopy, many cell biologists have turned to immunocytochemistry techniques, but fluorescence labeling remains the foundation of these technologies. Therefore, electron microscopy observation remains an indispensable method, and its application in biology continues to expand with the development of new devices and techniques.

Low-Vacuum Scanning Electron Microscopy (LVSEM) allows high-resolution imaging of non-conductive samples by neutralizing the negative charge accumulated on non-conductive materials with positive ions in residual gas molecules. However, traditional metal staining methods require the use of uranium compounds, which are not only harmful to operators but also limit the widespread application of LVSEM. Therefore, developing a safe, rapid, and uranium-free metal staining method has become an important research goal.

Source of the Paper

This paper was co-authored by Akira Sawaguchi, Takeshi Kamimura, Kyoko Kitagawa, Yoko Nagashima, and Nobuyasu Takahashi from the Division of Ultrastructural Cell Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, and Hitachi High-Tech Corporation. The paper was published in 2024 in the journal npj Imaging under the title “KMnO₄/Pb Staining Allows Uranium-Free Imaging of Tissue Architectures in Low Vacuum Scanning Electron Microscopy.”

Research Process

1. Sample Preparation

The study used 10-week-old male Wistar rats, fixed by perfusion through the left ventricle with 10% formalin (4% paraformaldehyde) or a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde. Subsequently, organs such as the kidney, lung, trachea, and auricle were excised and further fixed for 2 hours, followed by dehydration and paraffin embedding using an automatic tissue processor. Finally, 5-micron or 20-micron thick paraffin sections were prepared.

2. Correlative Light Microscopy and LVSEM Imaging

Paraffin sections were deparaffinized and rehydrated, then stained with hematoxylin and eosin (H&E) and observed under a light microscope. After observation, the sections were incubated in xylene for 18-24 hours to remove the coverslips, followed by metal staining.

3. KMnO₄/Pb Metal Staining

Sections were first treated with 0.2% potassium permanganate (KMnO₄) for 5 minutes, followed by staining with Reynolds’ lead citrate solution (Pb) for 3 minutes. The optimal KMnO₄ concentration and treatment time were determined through grayscale histogram analysis.

4. LVSEM Imaging

Stained sections were observed under LVSEM (Hitachi High-Tech Corporation’s TM4000Plus II) at accelerating voltages of 5, 10, 15, or 20 kV. Montage images of the entire section were obtained using semi-automatic tiling techniques.

Main Results

1. Optimization of KMnO₄/Pb Staining

Through grayscale histogram analysis, the optimal KMnO₄ concentration was determined to be 0.2%, with a treatment time of 5 minutes. Compared to traditional uranium/lead (Ur/Pb) staining, KMnO₄/Pb staining showed similar staining effects and image contrast in tissues such as the glomerulus, uriniferous tubules, transitional epithelium of the ureter, and thyroid follicular epithelium.

2. Elemental Analysis

Elemental analysis revealed that the backscattered electron (BSE) signal intensity in both KMnO₄/Pb and Ur/Pb staining was primarily determined by the amount of lead (Pb) deposition. KMnO₄ oxidation enhanced Pb deposition, thereby increasing the BSE signal intensity.

3. Comparison with Other Uranium-Free Staining Methods

Compared to uranium-free staining methods such as platinum blue/lead (Pt-blue/Pb), oolong tea extract/lead (Ote/Pb), and samarium triacetate/lead (Sm/Pb), KMnO₄/Pb staining exhibited higher contrast in the internal elastic lamina of muscular arteries and the interterritorial matrix of elastic cartilage.

4. Three-Dimensional Imaging

KMnO₄/Pb staining applied to thick-section LVSEM successfully achieved three-dimensional imaging of tissues such as the glomerulus, uriniferous tubules, and lung bronchioles, revealing complex cell/tissue architectures.

Conclusion

This study developed a uranium-free KMnO₄/Pb metal staining method that enables multiscale imaging of paraffin sections under low-vacuum scanning electron microscopy. The method is not only safe and rapid but also provides image contrast equivalent to traditional Ur/Pb staining, making it suitable for ultrastructural observation. KMnO₄ oxidation enhanced Pb deposition, thereby increasing the BSE signal intensity, particularly showing unique staining effects in elastic tissues.

Research Highlights

1. Uranium-Free Staining Method: KMnO₄/Pb staining avoids the use of harmful uranium compounds, providing a new approach for the widespread application of biomedical electron microscopy.
2. Multiscale Imaging: From centimeter-scale light microscopy to nanometer-scale LVSEM imaging, comprehensive analysis of complex cell/tissue architectures was achieved.
3. Three-Dimensional Imaging: Thick-section LVSEM combined with KMnO₄/Pb staining successfully revealed the three-dimensional structure of cells/tissues.
4. User-Friendly: The method requires no special equipment or techniques and is suitable for routine pathological diagnosis and retrospective studies.

Significance and Value

This study provides a safe and efficient electron microscopy imaging method for biomedical research, particularly with important applications in histopathological diagnosis and three-dimensional structural analysis. Additionally, the method can be used for retrospective studies, enabling ultrastructural analysis of paraffin sections preserved for many years, further advancing biomedical research.

By developing a uranium-free KMnO₄/Pb staining method, this study not only addresses the safety issues of traditional staining methods but also opens new avenues for the widespread application of electron microscopy in the biomedical field.