Circular intensity differential scattering (CIDS) scanning microscopy to image chromatin-DNA nuclear organization

Gratiet, Aymeric Le; Pesce, Luca; Oneto, Michele; Marongiu, Riccardo; Zanini, Giulia; Bianchini, Paolo; Diaspro, Alberto

doi:10.1364/osac.1.001068

Cited by 24 publications

(34 citation statements)

References 28 publications

(40 reference statements)

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“…From longer nucleosome arrays to chromatin fiber, other super-resolution techniques, such as Photoactivated Localization Microscopy (PALM) have been used to extrapolate chromatin topology in FIGURE 3 | As with modeling approaches, in experiments different techniques are required to study different orders of magnitude in chromatin: (A) NCP imaged with Cryo-Em (adapted from Kobayashi et al, 2019), (B) NCPs with histone tails AFM image (Filenko et al, 2012), (C) Nucleosome array, AFM image (adapted from Krzemien et al, 2017), (D) Isolated Hek nucleus imaged with CIDS (a), labeled with Hoechst for chromatin-DNA organization imaging. The fluorescence labeling (b) is used as a fingerprint of chromatin to demonstrate the correlation with the label-free approach using circular polarization excitation (Le Gratiet et al, 2018).…”

Section: Experimental Studies Of Chromatin: From the Nucleosome To Thmentioning

confidence: 99%

“…the nucleus from nucleosome dynamics. Label-free techniques are also used to study chromatin at the nuclear level, such as Circular Intensity Differential Scanning (CIDS) in Le Gratiet et al (2018). In this work, it is shown that the main advantage of this polarimetric method compared to standard fluorescence microscopy is the capability to obtain specific contrast mechanisms due to the chiral organization of the DNA in a label-free approach without a priori knowledge of the sample.…”

Section: Experimental Studies Of Chromatin: From the Nucleosome To Thmentioning

confidence: 99%

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Chromatin Compaction Multiscale Modeling: A Complex Synergy Between Theory, Simulation, and Experiment

Bendandi

Dante

Zia

et al. 2020

Front. Mol. Biosci.

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Understanding the mechanisms that trigger chromatin compaction, its patterns, and the factors they depend on, is a fundamental and still open question in Biology. Chromatin compacts and reinforces DNA and is a stable but dynamic structure, to make DNA accessible to proteins. In recent years, computational advances have provided larger amounts of data and have made large-scale simulations more viable. Experimental techniques for the extraction and reconstitution of chromatin fibers have improved, reinvigorating theoretical and experimental interest in the topic and stimulating debate on points previously considered as certainties regarding chromatin. A great assortment of approaches has emerged, from all-atom single-nucleosome or oligonucleosome simulations to various degrees of coarse graining, to polymer models, to fractal-like structures and purely topological models. Different fiber-start patterns have been studied in theory and experiment, as well as different linker DNA lengths. DNA is a highly charged macromolecule, making ionic and electrostatic interactions extremely important for chromatin topology and dynamics. Indeed, the repercussions of varying ionic concentration have been extensively examined at the computational level, using all-atom, coarse-grained, and continuum techniques. The presence of high-curvature AT-rich segments in DNA can cause conformational variations, attesting to the fact that the role of DNA is both structural and electrostatic. There have been some tentative attempts to describe the force fields governing chromatin conformational changes and the energy landscapes of these transitions, but the intricacy of the system has hampered reaching a consensus. The study of chromatin conformations is an intrinsically multiscale topic, influenced by a wide range of biological and physical interactions, spanning from the atomic to the chromosome level. Therefore, powerful modeling techniques and carefully planned experiments are required for an overview of the most relevant phenomena and interactions. The topic provides fertile ground for interdisciplinary studies featuring a synergy between theoretical and experimental scientists from different fields and the cross-validation of respective results, with a multi-scale perspective. Here, we summarize some of the most representative approaches, and focus on the importance of electrostatics and solvation, often overlooked aspects of chromatin modeling.

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Section: Experimental Studies Of Chromatin: From the Nucleosome To Thmentioning

confidence: 99%

Section: Experimental Studies Of Chromatin: From the Nucleosome To Thmentioning

confidence: 99%

Chromatin Compaction Multiscale Modeling: A Complex Synergy Between Theory, Simulation, and Experiment

Bendandi

Dante

Zia

et al. 2020

Front. Mol. Biosci.

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“…Due to the flexibility of this method and its local sensitivity to optical anisotropy at the nanoscale level, CD/CIDS scanning microscopy has been demonstrated recently as an important tool for label-free imaging in a large range of applications such as for metamaterials characterization [ 133 , 134 , 135 , 136 , 137 ] or chiral polymers organization under certain constraints [ 129 , 138 , 139 ]. Finally, the versatility of this method has shown its potentiality for multimodal imaging of chromatin characterization proposed recently by our group [ 130 ]. We implemented a CIDS imaging modality into a commercial scanning confocal fluorescence microscope in order to observe isolated HEK cell nuclei that were marked with Hoechst 33342, a chromatin binding fluorophore.…”

Section: Cids Microscopy Configurationmentioning

confidence: 99%

“…( b ) Optical microscope using one photoelastic modulator synchronized with a lock-in amplifier and an XY translating sample holder, inspired by [ 100 , 126 , 127 ]. ( c ) Optical scanning microscope using a photoelastic modulator synchronized with a lock-in amplifier, inspired by [ 128 , 129 , 130 ]. PC: Pockels Cell.…”

Section: Figurementioning

confidence: 99%

Circular Intensity Differential Scattering for Label-Free Chromatin Characterization: A Review for Optical Microscopy

2020

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Circular Intensity Differential Scattering (CIDS) provides a differential measurement of the circular right and left polarized light and has been proven to be a gold standard label-free technique to study the molecular conformation of complex biopolymers, such as chromatin. In early works, it has been shown that the scattering component of the CIDS signal gives information from the long-range chiral organization on a scale down to 1/10th–1/20th of the excitation wavelength, leading to information related to the structure and orientation of biopolymers in situ at the nanoscale. In this paper, we review the typical methods and technologies employed for measuring this signal coming from complex macro-molecules ordering. Additionally, we include a general description of the experimental architectures employed for spectroscopic CIDS measurements, angular or spectral, and of the most recent advances in the field of optical imaging microscopy, allowing a visualization of the chromatin organization in situ.

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“…From longer nucleosome arrays to chromatin fibre, other super-resolution techniques, such as Photoactivated Localization Microscopy (PALM) have been used to extrapolate chromatin topology in the nucleus from nucleosome dynamics. Label-free techniques are also used to study chromatin at the nuclear level, such as Circular Intensity Differential Scanning (CIDS) (51). SAXS and Cryo-EM have also been used in structural analysis of the fibre up to the chromosome level (52)(53)(54)(55).…”

Section: Experimental Studies Of Chromatin: From the Nucleosome To Thmentioning

confidence: 99%

Electrostatics and Solvation: Essential Determinants of Chromatin Compaction

Bendandi

Dante²,

Zia³

et al. 2019

Preprint

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Chromatin compaction is a process of fundamental importance in Biology, as it greatly influences cellular function and gene expression. The dynamics of compaction is determined by the interactions between DNA and histones, which are mainly mechanical and electrostatic. The high charge of DNA makes electrostatics extremely important for chromatin topology and dynamics. Besides their mechanical and steric role in the chromatin fibre, linker DNA length and linker histone presence and binding position also bear great electrostatic consequences. Electrostatics in chromatin is also indirectly linked to the DNA sequence: the presence of high-curvature AT-rich segments in DNA can cause conformational variations with electrostatic repercussions, attesting to the fact that the role of DNA is both structural and electrostatic. Electrostatics in this system has been analysed by extensively examining at the computational level the repercussions of varying ionic concentration, using all-atom, coarse-grained, and continuum models. There have been some tentative attempts to describe the force fields governing chromatin conformational changes and the energy landscapes of these transitions, but the intricacy of the system has hampered reaching a consensus. Chromatin compaction is a very complex issue, depending on many factors and spanning orders of magnitude in space and time in its dynamics. Therefore, comparison and complementation of theoretical models with experimental results is fundamental. Here, we present existing approaches to analyse electrostatics in chromatin and the different points of view from which this issue is treated. We pay particular attention to solvation, often overlooked in chromatin studies. We also present some numerical results on the solvation of nucleosome core particles. We discuss experimental techniques that have been combined with computational approaches and present some related experimental data such as the Z-potential of nucleosomes at varying ionic concentrations. Finally, we discuss how these observations support the importance of electrostatics and solvation in chromatin models. SIGNIFICANCE This work explores the determinants of chromatin compaction, focusing on the importance of electrostatic interactions and solvation. Chromatin compaction is an intrinsically multiscale issue, since processes concerning chromatin occur on a wide range of spatial and temporal scales. Since DNA is a highly charged macromolecule, electrostatic interactions are extremely significant for chromatin compaction, an effect examined in this work from many angles, such as the importance of ionic concentration and different ionic types, DNA-protein interactions, and solvation. Solvation is often overlooked in chromatin studies, especially in coarse-grained models, where the nucleosome core, the building block of the chromatin fibre, is represented as a rigid body, even though it has been observed that solvation influences chromatin even at the base-pair level.

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Circular intensity differential scattering (CIDS) scanning microscopy to image chromatin-DNA nuclear organization

Cited by 24 publications

References 28 publications

Chromatin Compaction Multiscale Modeling: A Complex Synergy Between Theory, Simulation, and Experiment

Chromatin Compaction Multiscale Modeling: A Complex Synergy Between Theory, Simulation, and Experiment

Circular Intensity Differential Scattering for Label-Free Chromatin Characterization: A Review for Optical Microscopy

Electrostatics and Solvation: Essential Determinants of Chromatin Compaction

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