Microscopy methods for the in vivo study of nanoscale nuclear organization

Eukaryotic genomes are highly compacted within the nucleus and organized into complex 3D structures across various genomic and physical scales. Organization within the nucleus plays a key role in gene regulation, both facilitating regulatory interactions to promote transcription while also enabling the silencing of other genes. Despite the functional importance of genome organization in determining cell identity and function, investigating nuclear organization across this wide range of physical scales has been challenging. Microscopy provides the opportunity for direct visualization of nuclear structures and has pioneered key discoveries in this field. Nonetheless, visualization of nanoscale structures within the nucleus, such as nucleosomes and chromatin loops, requires super-resolution imaging to go beyond the ~220 nm diffraction limit. Here, we review recent advances in imaging technology and their promise to uncover new insights into the organization of the nucleus at the nanoscale. We discuss different imaging modalities and how they have been applied to the nucleus, with a focus on super-resolution light microscopy and its application to in vivo systems. Finally, we conclude with our perspective on how continued technical innovations in super-resolution imaging in the nucleus will advance our understanding of genome structure and function.