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Updated: Aug 16, 2026
Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification
Published on: November 15, 2010
Visualization of DNA within mitochondria by osmium-ammine staining of mouse duodenal crypt cells
D F Liu1, M el-Alfy, C P Leblond
1Department of Anatomy, McGill University, Montreal, Quebec, Canada.
Abstract:
Previous investigators have examined mitochondrial DNA (mtDNA) in the electron microscope (EM) after extraction from mitochondria and rotary shadowing. We have observed mtDNA in situ by the osmiumammine procedure for specific staining of DNA in the EM. The procedure was modified to improve the regularity of the staining and then applied to the rapidly dividing cells present in mouse duodenal crypts. In the stained sections of these cells, 25% of the mitochondria exhibited discrete reactive filaments. The filaments, whether observed directly or in stereopairs, appeared either irregular or arranged into distinct patterns, some of which were similar to those previously described after rotary shadowing of duplicating mtDNA: namely, simple and double circular figures, displacement loops and supercoiled forms. The filaments could be traced in serial sections of the same mitochondria and, therefore, were not artifacts. Moreover, their disappearance after DNase digestion demonstrated that they were composed of DNA. It is concluded that mtDNA can be visualized by the modified osmium-ammine technique and may show patterns that can be interpreted as phases in its replication.
Insights
Researchers visualized mitochondrial DNA (mtDNA) within cells using a novel staining technique. This method revealed mtDNA structures resembling replication intermediates, offering new insights into mitochondrial DNA replication.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Mitochondrial DNA (mtDNA) visualization has historically relied on extraction and rotary shadowing.
- Previous methods required isolating mtDNA from mitochondria, potentially altering its native structure.
Purpose of the Study:
- To develop and apply a technique for visualizing intact mtDNA within mitochondria in situ.
- To observe potential mtDNA replication intermediates within cellular structures.
Main Methods:
- Modified osmium-ammine staining procedure for specific DNA visualization in electron microscopy (EM).
- Application of the technique to rapidly dividing cells from mouse duodenal crypts.
- Analysis of stained mitochondria using EM, including serial sectioning and DNase digestion.
Main Results:
- Successfully visualized mtDNA filaments in situ within 25% of observed mitochondria.
- Observed mtDNA structures included circular figures, displacement loops, and supercoiled forms, consistent with replication intermediates.
- Filaments were confirmed as DNA through DNase digestion and serial section analysis, ruling out artifacts.
Conclusions:
- The modified osmium-ammine technique enables visualization of mtDNA in its native mitochondrial environment.
- Observed mtDNA configurations suggest potential in situ phases of mitochondrial DNA replication.
- This technique provides a valuable tool for studying mtDNA dynamics and replication.
