Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina
Mäntylä, E., Montonen, T., Azzari, L., Mattola, S., Hannula, M., Vihinen-Ranta, M., Hyttinen, J., Vippola, M., Foi, A., Nymark, S., & Ihalainen, T. O. (2023). Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina. Molecular Biology of the Cell, 34(9). https://doi.org/10.1091/mbc.e22-09-0448
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Molecular Biology of the CellAuthors
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2023Copyright
© 2023 Mäntylä et al. This article is distributed by The American Society for Cell
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Investigation of nuclear lamina architecture relies on super-resolved 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 super-resolution 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 3D-printed gel casting equipment. We show that in comparison to 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 super-resolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization - a prerequisite for studying intranuclear structural co-regulation of cell function and fate.
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American Society for Cell Biology (ASCB)ISSN Search the Publication Forum
1059-1524Publication in research information system
https://converis.jyu.fi/converis/portal/detail/Publication/183849181
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Research Council of FinlandFunding program(s)
Academy Project, AoFAdditional information about funding
This work was supported by the Academy of Finland under the award numbers 308315 and 314106 (TOI), 330896 (MVR), 332615 (EM), and the Centre of Excellence in Body-on-Chip Research (312412 (JH)), 336357 (PROFI6 - TAU Imaging Research Platform (LA, MV, AF), and by the Jane and Aatos Erkko Foundation (MVR).License
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