Data segmentation and object rendering is required for localization super-resolution microscopy, fluorescent photoactivation localization microscopy (FPALM), and direct stochastic optical reconstruction microscopy (dSTORM). We developed and validated methods for segmenting objects based on Delaunay triangulation in 3D space, followed by facet culling. We applied them to visualize mitochondrial nucleoids, which confine DNA in complexes with mitochondrial (mt) transcription factor A (TFAM) and gene expression machinery proteins, such as mt single-stranded-DNA-binding protein (mtSSB). Eos2-conjugated TFAM visualized nucleoids in HepG2 cells, which was compared with dSTORM 3D-immunocytochemistry of TFAM, mtSSB, or DNA. The localized fluorophores of FPALM/dSTORM data were segmented using Delaunay triangulation into polyhedron models and by principal component analysis (PCA) into general PCA ellipsoids. The PCA ellipsoids were normalized to the smoothed volume of polyhedrons or by the net unsmoothed Delaunay volume and remodeled into rotational ellipsoids to obtain models, termed DVRE. The most frequent size of ellipsoid nucleoid model imaged via TFAM was 35 × 45 × 95 nm; or 35 × 45 × 75 nm for mtDNA cores; and 25 × 45 × 100 nm for nucleoids imaged via mtSSB. Nucleoids encompassed different point density and wide size ranges, speculatively due to different activity stemming from different TFAM/mtDNA stoichiometry/density. Considering twofold lower axial vs. lateral resolution, only bulky DVRE models with an aspect ratio >3 and tilted toward the xy-plane were considered as two proximal nucleoids, suspicious occurring after division following mtDNA replication. The existence of proximal nucleoids in mtDNA-dSTORM 3D images of mtDNA “doubling”-supported possible direct observations of mt nucleoid division after mtDNA replication.
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The authors thank Martin Bartoš (Alef, Ltd., Prague) for help with nucleoid modeling using the Delaunay algorithm and Paraview software; and to Prof. Daniel F. Bogenhagen (Department of Pharmacological Sciences, State University of New York at Stony Brook) for providing the Eos2 vector and rabbit anti-TFAM antibodies. The project was principally supported by a grant of the Grant Agency of the Czech Republic (GACR) No. 13-02033S to P.J.; by the research project RVO67985823 to the Institute of Physiology; and also by the project BIOCEV—Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund. The latter source was also co-funded by the European Social Fund and the state budget of the Czech Republic.
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Alán, L., Špaček, T. & Ježek, P. Delaunay algorithm and principal component analysis for 3D visualization of mitochondrial DNA nucleoids by Biplane FPALM/dSTORM. Eur Biophys J 45, 443–461 (2016). https://doi.org/10.1007/s00249-016-1114-5
- 3D object segmentation
- Delaunay algorithm
- Principal component analysis
- 3D super-resolution microscopy
- Mitochondrial DNA replication