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

Abstract

Investigation of nuclear lamina architecture relies on super–resolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules poses challenges within the molecularly crowded nucleus. We developed an iterative indirect immunofluorescence staining (IT–IF) approach combined with expansion microscopy (ExM) and structured illumination microscopy (SIM) improving super–resolution microscopy of subnuclear nanostructures like nuclear lamina. First, we prove that ExM is applicable in analyzing structurally highly compacted nuclear multiprotein complexes such as viral nucleocapsids. Second, we provide technical improvements to the ExM method, including 3D–printed gel casting equipment. Third, show that in comparison to conventional immunostaining, iterative labeling of intranuclear targets like nuclear lamina results in a higher signal-to-background –ratio (SBR) and mean fluorescence intensity and use structured illumination microscopy and ExM to critically point out that iterative immunostaining results in improved target labeling density, benefiting the structural analyses via ExM. Lastly, we develop a signal processing pipeline for noise estimation, denoising and deblurring in analysis of lamin network and provide this platform to the microscopy imaging research community. Our work reveals the potential of signal-resolved IT–IF in super-resolution imaging of lamin network organization —a prerequisite for studying intranuclear structural co-regulation of cell function and fate.