A user-interactive algorithm quantifying nuclear pore complex distribution within the nuclear lamina network in single molecular localization microscopic image

• Published in: Methods (San Diego, Calif.) Vol. 157; pp. 42 - 46
• Main Authors: Lim, John S.Y., Wright, Graham D., Burke, Brian, Xie, Wei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.2019
• Subjects: algorithms; data collection; Fiji; fluorescence microscopy; mammals; Nuclear envelope; Nuclear lamin; nuclear lamina; nuclear pore; Nuclear pore complex; quantitative analysis; somatic cells; Super-resolution microscopy; Super-resolution microscopy; Fiji; Nuclear pore complex; Nuclear envelope; Nuclear lamin
• ISSN: 1046-2023; 1095-9130; 1095-9130
• DOI: 10.1016/j.ymeth.2018.09.006

•The nuclear envelope resists quantitative study by conventional light microscopy.•Super-resolution images contain sufficient data to allow precise numerical analysis.•An algorithm to quantify nuclear pore complex and lamina distribution is described.•The algorithm can be easily adapted to the analysis of other cellular components. For decades, components of the mammalian nuclear envelope (NE), such as the nuclear lamina and nuclear pore complexes (NPCs), have been largely resistant to quantitative cell biological analysis using conventional fluorescence microscopy. This is in part due to their sub diffraction limit dimensions. Super-resolution microscopy, a major advancement in cell biology research, has now made possible the acquisition of images in which nuclear lamin networks and single NPCs can be resolved in intact mammalian somatic cells. In particular, single molecule localization microscopy is able to generate data sets that are accurate enough to allow detailed quantitative analysis. Here we describe an algorithm that will identify the centroid of single NPCs and will determine their localization relative to the distribution of lamin protein filaments. Using this algorithm, a percentage of NPCs localized within the nuclear lamin network was accurately calculated, that could be compared between cells expressing different lamin complements. With modifications tweaked according to user specified sample images, this algorithm serves as a semi-automatic and fast computational tool to quantify and compare the localization and distribution of two or more cellular components at the nanometre scale.