1. Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy
- Author
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Oneto, Michele, Scipioni, Lorenzo, Sarmento, Maria J, Cainero, Isotta, Pelicci, Simone, Furia, Laura, Pelicci, Pier G, Dellino, Gaetano I, Bianchini, Paolo, Faretta, Mario, Gratton, Enrico, Diaspro, Alberto, and Lanzanò, Luca
- Subjects
Bioengineering ,Genetics ,Nanotechnology ,1.1 Normal biological development and functioning ,Underpinning research ,Generic health relevance ,Cell Nucleus ,Color ,Humans ,MCF-7 Cells ,Microscopy ,Spectrum Analysis ,Physical Sciences ,Chemical Sciences ,Biological Sciences ,Biophysics - Abstract
Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus.
- Published
- 2019