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

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. (Words: 175) ### Competing Interest Statement The authors have declared no competing interest. * ExM : Expansion microscopy FWHM : Full width at half maximum HSV-1 : Herpes simplex virus 1 IT-IF : Iterative indirect immunofluorescence staining LA/C-C : Lamin A/C C-terminus LA/C-N : Lamin A/C N-terminus LA/C-rod : Lamin A/C rod-domain NE : Nuclear envelope PAA : Polyacrylamide SBR : Signal-to-background-ratio SIM : Structured illumination microscopy SR-SIM : Super-resolution structured illumination microscopy