Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

Abstract

2Deciphering the spatiotemporal coordination between nuclear functions is important to 3 understand its role in the maintenance of human genome. In this context, superresolution 4 microscopy has gained considerable interest as it can be used to probe the spatial organization of 5 namely the ensemble of microscopy techniques providing optical resolution below the diffraction 59 limit (12). For instance, in single molecule localization microscopy (SMLM), the fluorophores are 60 sequentially switched on and off and localized with high precision on each frame, resulting in 61 images with a resolution down to ~20 nm (13)(14)(15). In stimulated emission depletion (STED) 62 microscopy the fluorophores are selectively switched off at the periphery of the diffraction-limited 63 detection spot by a second, doughnut-shaped laser beam, producing an immediate improvement of 64 spatial resolution down to ~50 nm (16). Thus, superresolution can be used to probe spatial 65 organization in the 20-250 nm range, which corresponds to the range of higher order organization of 66 chromatin in the nucleus. This nanoscale spatial organization can be studied in a 'static' way, from 67 images of fixed cells or in a 'dynamic' way, from acquisition of superresolution data in live cells. 68Methods that provide a quantitative measure of colocalization from static multicolor 69 superresolution images can be divided in two major groups: object-based methods, which perform a 70 segmentation of the image into objects prior to analyzing their relative spatial distributions, and 71 pixel-based methods, which extract correlation coefficients from pixel intensities (17, 18). In 72 general, object-based analysis can be performed on any type of superresolution image as long as the 73 target objects are well resolved and identified. In particular, object-based methods are often the 74 methods of choice in SMLM, where the acquired data are already segmented into a list of x y 75 coordinates of individual molecules (19-23). In principle, object-based analysis provides a full 76 description of the spatial distribution of the two labeled species: in fact, knowledge of the objects 77 coordinates allows (i) mapping the locations of the specimen with a higher level of proximity 78 and (ii) performing a statistical analysis of the relative distance between the particles. On the 79 other hand, a great advantage of pixel-based methods is that they do not require a pre-segmentation 80 of the images into objects but rely on the calculation of coefficients from the pixel intensity values 81 (24, 25). Pixel-based methods are routinely applied to quantify spatial distribution in multicolor 82 superresolution images (26-28). 83A method to study interactions in a 'dynamic' way is Fluorescence Cross-Correlation 84 Spectroscopy (FCCS), the dual channel version of Fluorescence Correlation Spectroscopy (FCS) 85 (29). In FCCS, the fraction of interacting particles is extracted from the analysis of temporal 86 intensity fluctuations originating from changes in fluorophore conc...