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Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina
Investigation of nuclear lamina architecture relies on superresolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staini...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10398900/ https://www.ncbi.nlm.nih.gov/pubmed/37342871 http://dx.doi.org/10.1091/mbc.E22-09-0448 |
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author | Mäntylä, Elina Montonen, Toni Azzari, Lucio Mattola, Salla Hannula, Markus Vihinen-Ranta, Maija Hyttinen, Jari Vippola, Minnamari Foi, Alessandro Nymark, Soile Ihalainen, Teemu O. |
author_facet | Mäntylä, Elina Montonen, Toni Azzari, Lucio Mattola, Salla Hannula, Markus Vihinen-Ranta, Maija Hyttinen, Jari Vippola, Minnamari Foi, Alessandro Nymark, Soile Ihalainen, Teemu O. |
author_sort | Mäntylä, Elina |
collection | PubMed |
description | Investigation of nuclear lamina architecture relies on superresolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staining approach combined with expansion microscopy (ExM) and structured illumination microscopy to improve superresolution microscopy of subnuclear nanostructures like lamins. We prove that ExM is applicable in analyzing highly compacted nuclear multiprotein complexes such as viral capsids and provide technical improvements to ExM method including three-dimensional–printed gel casting equipment. We show that in comparison with conventional immunostaining, IT-IF results in a higher signal-to-background ratio and a mean fluorescence intensity by improving the labeling density. Moreover, we present a signal-processing pipeline for noise estimation, denoising, and deblurring to aid in quantitative image analyses and provide this platform for the microscopy imaging community. Finally, we show the potential of signal-resolved IT-IF in quantitative superresolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization—a prerequisite for studying intranuclear structural coregulation of cell function and fate. |
format | Online Article Text |
id | pubmed-10398900 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-103989002023-10-16 Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina Mäntylä, Elina Montonen, Toni Azzari, Lucio Mattola, Salla Hannula, Markus Vihinen-Ranta, Maija Hyttinen, Jari Vippola, Minnamari Foi, Alessandro Nymark, Soile Ihalainen, Teemu O. Mol Biol Cell Special Issue on Quantitative Cell Biology Investigation of nuclear lamina architecture relies on superresolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staining approach combined with expansion microscopy (ExM) and structured illumination microscopy to improve superresolution microscopy of subnuclear nanostructures like lamins. We prove that ExM is applicable in analyzing highly compacted nuclear multiprotein complexes such as viral capsids and provide technical improvements to ExM method including three-dimensional–printed gel casting equipment. We show that in comparison with conventional immunostaining, IT-IF results in a higher signal-to-background ratio and a mean fluorescence intensity by improving the labeling density. Moreover, we present a signal-processing pipeline for noise estimation, denoising, and deblurring to aid in quantitative image analyses and provide this platform for the microscopy imaging community. Finally, we show the potential of signal-resolved IT-IF in quantitative superresolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization—a prerequisite for studying intranuclear structural coregulation of cell function and fate. The American Society for Cell Biology 2023-08-01 /pmc/articles/PMC10398900/ /pubmed/37342871 http://dx.doi.org/10.1091/mbc.E22-09-0448 Text en © 2023 Mäntylä et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. https://creativecommons.org/licenses/by-nc-sa/4.0/This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial-Share Alike 4.0 International Creative Commons License. |
spellingShingle | Special Issue on Quantitative Cell Biology Mäntylä, Elina Montonen, Toni Azzari, Lucio Mattola, Salla Hannula, Markus Vihinen-Ranta, Maija Hyttinen, Jari Vippola, Minnamari Foi, Alessandro Nymark, Soile Ihalainen, Teemu O. Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina |
title | Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina |
title_full | Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina |
title_fullStr | Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina |
title_full_unstemmed | Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina |
title_short | Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina |
title_sort | iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina |
topic | Special Issue on Quantitative Cell Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10398900/ https://www.ncbi.nlm.nih.gov/pubmed/37342871 http://dx.doi.org/10.1091/mbc.E22-09-0448 |
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