Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina: Brief report

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Abstract

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.

Original languageEnglish
Article numbermbcE22090448
Number of pages13
JournalMolecular Biology of the Cell
Volume34
Issue number9
Early online date21 Jun 2023
DOIs
Publication statusPublished - Aug 2023
Publication typeA1 Journal article-refereed

Publication forum classification

  • Publication forum level 2

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