Imaging Nuclear Envelopes Using Correlative AFM/Fluorescence Microscopy.

The nuclear envelope (NE) is the hallmark of eukaryotic cells. It is a complex structure composed of two concentric lipid bilayers separated by a 50 nm lumen. While the outer nuclear membrane is continuous with the endoplasmic reticulum, the inner nuclear membrane contains a unique set of proteins. Methods have been developed several decades ago to prepare NEs from Xenopus laevis oocytes, exposing inner and outer nuclear membranes, which makes them ideal samples for probe scanning methods such as atomic force microscopy (AFM). In addition, biochemical preparations of human NEs have been valuable tools to investigate the structure of their nuclear pore complexes (NPCs). However, recent data have highlighted that membrane tension loss during NE preparation changes the morphology of NPCs. In this chapter, we outline a novel approach to extract nuclei from cultured mammalian cells, affix them onto glass substrates, and gently open them, thereby maintaining membrane tension and preserving NPC structure for subsequent AFM imaging of both inner and outer nuclear membranes. Furthermore, we detail a protocol for immuno-labeling and correlative imaging by AFM/dSTORM (direct Stochastic Optical Reconstruction Microscopy). Additionally, we provide comprehensive guidance on data analysis, in particular for proper correlation of the optical and AFM images. Notably, the sample preparation steps only require basic materials. The procedures described here could be easily extended to other cellular models such as plant cells or cells isolated from animal tissues.