Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures

Sarmento, Maria J.; Oneto, Michele; Pelicci, Simone; Pesce, Luca; Scipioni, Lorenzo; Faretta, Mario; Furia, Laura; Dellino, Gaetano Ivan; Pelicci, Pier Giuseppe; Bianchini, Paolo; Diaspro, Alberto; Lanzanò, Luca

doi:10.1038/s41467-018-05963-2

Cited by 46 publications

(50 citation statements)

References 44 publications

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“…To compare STED-ICCS with object-based analysis, we simulated dual color images of nuclear foci at variable densities (Fig.1). In each channel, the nuclear foci were simulated as N point-like particles distributed in a 16 µm wide circular region (40) and convoluted with a Gaussian Point Spread Function (PSF) with a Full Width Half Maximum (FWHM) of 3 pixels, corresponding to 120 nm. We simulated distributions of foci randomly distributed in each channel (uncorrelated), with a fraction f coloc =0.25 of foci colocalizing in the two channels (colocalized) and with a fraction f coloc =0.25 of foci colocalizing only in a specific sub-portion of the image (colocalized in a zone).…”

Section: Resultsmentioning

confidence: 99%

“…For instance, Structured illumination Microscopy (SIM), and its point-scanning equivalent Image Scanning Microscopy (ISM), are superresolution techniques compatible with live cell imaging, even if their resolution improvement is limited to a factor of ~2 (12). The same STED nanoscopy includes several variants developed to reduce the STED beam intensity and its potentially photo-damaging effects (40, 52, 53). Thus, we expect that our ICCS formalism could be applied to live cell super-resolved images obtained, for instance, by dual color STED- or SIM-based setups.…”

Section: Discussionmentioning

confidence: 99%

“…Dual color images of nuclear foci were simulated using MATLAB. For each channel, the object consisted in a variable number N of point-like emitters distributed randomly inside a circular area with a diameter of 16 µm (40). The images were made by 512×512 pixels with a pixel size of 40 nm.…”

Section: Methodsmentioning

confidence: 99%

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Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

Oneto

Scipioni

Sarmento

et al. 2019

Preprint

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

Oneto

Scipioni

Sarmento

et al. 2019

Preprint

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“…Indeed, FCS can be combined with STED microscopy (STED-FCS) to perform fluctuation analysis on sub-diffraction observation volumes (47). In STED, the size of observation volume can be easily tuned by changing the depletion power (48). Thus, an important aspect of STED-FCS is the capability to probe diffusion at different sub-diffraction spatial scales, i.e.…”

Section: Resultsmentioning

confidence: 99%

Measuring mobility in chromatin by intensity sorted FCS

Bona

Mancini

Mazza

et al. 2018

Preprint

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6The architectural organization of chromatin can play an important role in genome regulation 7 by affecting the mobility of molecules within its surroundings via binding interactions and 8 molecular crowding. The diffusion of molecules at specific locations in the nucleus can be 9 studied by Fluorescence Correlation Spectroscopy (FCS), a well-established technique based 10 on the analysis of fluorescence intensity fluctuations detected in a confocal observation 11 volume. However, detecting subtle variations of mobility between different chromatin regions 12 remains challenging with currently-available FCS methods. 13 Here we introduce a method that samples multiple positions by slowly scanning the FCS 14 observation volume across the nucleus. Analyzing the data in short time segments, we 15 preserve the high temporal resolution of single-point FCS while probing different nuclear 16 regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a 17 reference, we efficiently sort the FCS segments into different populations and obtain average 18 correlation functions that are associated to different chromatin regions. This sorting and 19 averaging strategy renders the method statistically robust while preserving the observation of 20 intranuclear variations of mobility. 21Using this approach, we quantified diffusion of monomeric GFP in high versus low 22 chromatin density regions. We found that GFP mobility was reduced in heterochromatin, 23 especially within perinucleolar heterochromatin. Moreover, we found that modulation of 24 chromatin compaction by ATP depletion, or treatment with solution of different osmolarity, 25 differentially-affected the ratio of diffusion in both regions. Then, we used the approach to 26 probe the mobility of estrogen receptor-α (ER) in the vicinity of an integrated multicopy 27 prolactin gene array. Finally, we discussed the coupling of this method with stimulated 28 emission depletion (STED)-FCS, for performing FCS at sub-diffraction spatial scales. 29 30 31 32 33 34 74data at multiple observation volumes (25, 26). Another method that has been used to measure 75 fluctuations at, and between, different points in the nucleus is scanning FCS, which is easily 76 implemented on confocal laser scanning microscopes, but whose temporal resolution is 77 typically limited, by scanning, to the millisecond range (27, 28). Finally, diffusion maps have 78 been recently obtained from the analysis of raster image correlation spectroscopy (RICS) data 79 (29). However, these methods will only provide an accurate description of the mobility 80 properties in different nuclear regions if they are immobile during FCS data acquisition. 81A significant advantage can be gained if the different chromatin regions are identified with 82 the help of a reference marker (30). For instance, the intensity of a fluorescent protein can be 83 used to identify specific sub-nuclear regions or, simply, the intensity of a DNA marker (e.g. 84Hoechst) can be used to identify regions of different ch...

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“…Heterochromatin was first defined as the fraction of chromatin that remains condensed after mitosis while euchromatin has been described as low density, relatively decompacted chromatin, which includes mostly active regions rich in genes and regulatory sequences . The recent development of the so‐called super‐resolution fluorescence microscopy (SRM) techniques, including stimulated emission depletion (STED) microscopy , structured illumination microscopy (SIM) , and localization microscopy, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) , have extended the ultimate resolving power of optical microscopy far beyond the diffraction limit, facilitating access to the organization of chromatin at the nanoscale by optical means . For instance, STORM has been used to visualize chromatin higher‐order organization in single cell nuclei, revealing that both nucleosomes and DNA associate in heterogeneous nanodomains , and that distinct epigenetic states have a different nanoscale chromatin architecture .…”

Section: Introductionmentioning

confidence: 99%

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

Pelicci

Diaspro

Lanzanò

2019

Journal of Biophotonics

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@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.

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Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures

Cited by 46 publications

References 44 publications

Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

Measuring mobility in chromatin by intensity sorted FCS

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

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