title: Delaunay algorithm and principal component analysis for 3D visualization of mitochondrial DNA nucleoids by Biplane FPALM/dSTORM.
abstract: 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.
authors: Department of Membrane Transport Biophysics, No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors: Lukáš
authors: L
authors: Alán
authors: Department of Membrane Transport Biophysics, No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors: Tomáš
authors: T
authors: Špaček
authors: Department of Membrane Transport Biophysics, No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220, Prague 4, Czech Republic. jezek@biomed.cas.cz.
authors: Petr
authors: P
authors: Ježek
mesh_terms: D000465:Algorithms; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D025341:Principal Component Analysis
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C504091:NABP2 protein, human
keywords: 3D object segmentation; 3D super-resolution microscopy; Delaunay algorithm; Mitochondrial DNA replication; Nucleoids; Principal component analysis