Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.

@Chromatin nanoscale architecture in live cells can be studied by Förster resonance energy transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system, the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor‐to‐donor ratio rather than distance. Here, to get a FRET level independent of the acceptor‐to‐donor ratio, we combine fluorescence lifetime imaging detection of FRET with a normalization of the FRET level to a pixel‐wise estimation of the acceptor‐to‐donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that this acceptor‐to‐donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser‐induced microirradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.

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.

The SPLIT approach is a simple and efficient way to improve the spatial resolution of a super-resolved STED multi-dimensional image, i.e. a STED image in which an additional dimension encodes spatial information. Recently, we have demonstrated that the SPLIT can be applied to multidimensional STED images obtained with tunable depletion power. In this SPLIT-STED implementation, the additional dimension is represented by the depletion power, a parameter that can be easily tuned on any STED microscope. In this work, we introduce a modified implementation in which we tune also the excitation power. The tuning of the excitation power is used to modulate the number of photons collected for each STED image. We show that the modified SPLIT-STED method produces an improvement of spatial resolution for very different tuning patterns of the excitation intensity, improving the versatility of the SPLIT-STED approach. Interestingly, we find that the extent of photobleaching can be modulated by the excitation pattern, as it depends on the simultaneous impact of high STED and excitation powers. Thus, the tuning of the excitation power can improve applicability of the method to live cell imaging, potentially minimizing the photobleaching of the fluorophores and the phototoxicity on the biological samples during a SPLIT-STED acquisition. We apply the modified SPLIT-STED method to super-resolution imaging of nuclear periphery, in both fixed and live cells.

Super Resolution Microscopy revolutionized the approach to the study of molecular interactions by providing new quantitative tools to describe the scale below 100 nanometers. Single Molecule Localization Microscopy (SMLM) reaches a spatial resolution less than 50 nm with a precision in calculating molecule coordinates between 10 and 20 nanometers. However new procedures are required to analyze data from the list of molecular coordinates created by SMLM. We propose new tools based on Image Cross Correlation Spectroscopy (ICCS) to quantify the colocalization of fluorescent signals at single molecule level. These analysis procedures have been inserted into an experimental pipeline to optimize the produced results. We show that Fluorescent NanoDiamonds targeted to an intracellular compartment can be employed (i) to correct spatial drift to maximize the localization precision and (ii) to register confocal and SMLM images in correlative multiresolution, multimodal imaging. We validated the ICCS based approach on defined biological control samples and showed its ability to quantitatively map area of interactions inside the cell. The produced results show that the ICCS analysis is an efficient tool to measure relative spatial distribution of different molecular species at the nanoscale.