Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps

Scipioni, Lorenzo; Bona, Melody Di; Vicidomini, Giuseppe; Diaspro, Alberto; Lanzanò, Luca

doi:10.1038/s42003-017-0010-6

Cited by 41 publications

(36 citation statements)

References 51 publications

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“…ICCS performed on small sub-regions of the image (Fig.1e, h). It has been previously shown that, if the spatial correlation function is calculated locally, one can get maps of physical parameters such as protein velocity (43), particle size (44) or diffusion coefficient of a probe (45, 46). Local ICCS can be used to generate a map of the value of the colocalized fraction extracted by fitting the local auto- and cross-correlation functions (Fig.1e).…”

Section: Resultsmentioning

confidence: 99%

Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

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Scipioni

Sarmento

et al. 2019

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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%

Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

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Scipioni

Sarmento

et al. 2019

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“…As a validation of the method, we first measured differences in the diffusion coefficient of GFP in the nucleolus and the nucleoplasm of HeLa cells. It has been previously shown that even for a small inert probe like GFP, there is a clear difference in the values of the diffusion coefficient measured in the nucleoplasm in respect to the nucleolus (26, 29, 42). In this case, we used the GFP intensity level as a reference marker to distinguish the two nuclear regions.…”

Section: Resultsmentioning

confidence: 99%

“…Another method that has been used to measure fluctuations at, and between, different points in the nucleus is scanning FCS, which is easily implemented on confocal laser scanning microscopes, but whose temporal resolution is typically limited, by scanning, to the millisecond range (27, 28). Finally, diffusion maps have been recently obtained from the analysis of raster image correlation spectroscopy (RICS) data (29). However, these methods will only provide an accurate description of the mobility properties in different nuclear regions if they are immobile during FCS data acquisition.…”

Section: Introductionmentioning

confidence: 99%

Measuring mobility in chromatin by intensity sorted FCS

Bona

Mancini

Mazza

et al. 2018

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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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“…The study of protein dynamics and the use of EGFP as a probe for investigating the structure of cellular compartments have been used for decades 1,2,4,6,11,13,33,34 while, on a parallel effort, many microscopy techniques have been developed from the super-resolution imaging field (STED, ISM…) and from the fluorescence fluctuations field (pCF, iMSD, spot-variation FCS, N&B…) in order to provide increased spatiotemporal resolution to address dynamic as well as structural information of the cellular environment. Although many studies have focused on the coupling of STED with fluorescence fluctuation techniques, our work is, at the best of our knowledge, the first coupling ISM concepts to such techniques enabling, on a different spatial scale, the super-resolution investigation of many dynamic properties in a single analysis in a few seconds.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive correlation analysis for super-resolution dynamic fingerprinting of cellular compartments using the Zeiss Airyscan detector

et al. 2018

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The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.

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Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps

Cited by 41 publications

References 51 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

Comprehensive correlation analysis for super-resolution dynamic fingerprinting of cellular compartments using the Zeiss Airyscan detector

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