Ring Array Illumination Microscopy: Combination of Super-Resolution with Large Field of View Imaging and long Working Distances DOI Open Access

Johann von Hase,

Udo Birk, Bruno M. Humbel

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Sept. 6, 2023

Abstract Here we present a novel fluorescence microscopy concept which enables direct integration of Super-Resolution Microscopy (SRM) approaches (SIM/Nanosizing, STED, SMLM, MINFLUX, SIMFLUX) into systems with working distances (WD) up to the multicentimeter range while still allowing nanometer scale resolution at selected sites. This becomes possible by “synthetic aperture” illumination mode multiple, constructively interfering excitation beams positioned in “Ring-Array” arrangement around beam free interior zone containing instrumentation involved complementary imaging modes. The feasibility such correlative method is validated extensive numerical simulations; on basis these calculations, experimental implementation options are discussed. Such “Ring Array” modes may be useful for various methods, as combination light and electron same device (dCLEM); or low NA/large field-of-view widefield super-resolution sites (direct Correlative Opical Microscopy/dCOLM). Ring-Array supported will open perspectives variety disciplines, from material sciences biomedical applications.

Language: Английский

Quantitative Spatial Analysis of Chromatin Biomolecular Condensates using Cryo-Electron Tomography DOI Creative Commons
Huabin Zhou, Joshua Hutchings, Momoko Shiozaki

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 2, 2024

Phase separation is an important mechanism to generate certain biomolecular condensates and organize the cell interior. Condensate formation function remain incompletely understood due difficulties in visualizing condensate interior at high resolution. Here we analyzed structure of biochemically reconstituted chromatin through cryo-electron tomography. We found that traditional blotting methods sample preparation were inadequate, high-pressure freezing plus focused ion beam milling was essential maintain integrity. To identify densely packed molecules within condensate, integrated deep learning-based segmentation with novel context-aware template matching. Our approaches developed on condensates, also effective condensed regions situ native chromatin. Using these methods, determined average nucleosomes 6.1 12 Å resolution systems, respectively, have a nearly random orientation distribution both cases. should be applicable diverse some cells.

Language: Английский

Citations

3
Keeping Cells Alive in Microscopy DOI Creative Commons
Herbert Schneckenburger, Christoph Cremer

Biophysica, Journal Year: 2025, Volume and Issue: 5(1), P. 1 - 1

Published: Jan. 6, 2025

Light microscopy has emerged as one of the fundamental methods to analyze biological systems; novel techniques 3D and super-resolution (SRM) with an optical resolution down sub-nanometer range have recently been realized. However, most these achievements made fixed specimens, i.e., direct information about dynamics biosystem studied was not possible. This stimulated development live cell imaging approaches, including Low Illumination Fluorescence Microscopy, Sheet (Fluorescence) Microscopy (LSFM), or Structured (SIM). Here, we discuss perspectives, methods, relevant light doses advanced fluorescence keep cells alive at low levels phototoxicity.

Language: Английский

Citations

0
Linking DNA-packing density distribution and TAD boundary locations DOI Creative Commons
Luming Meng, Fu Kit Sheong, Qiong Luo

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(9)

Published: Feb. 25, 2025

DNA is heterogeneously packaged into chromatin, which further organized topologically associating domains (TADs) with sharp boundaries. These boundary locations are critical for genome regulation. Here, we explore how the distribution of DNA-packing density across chromatin affects TAD locations. We develop a polymer-physics-based model that utilizes accessibility data to parameterize along chromosomes, treating them as heteropolymers, and simulates stochastic folding these heteropolymers within nucleus yield conformation ensemble. Such an ensemble reproduces subset (over 60%) boundaries human genome, confirmed by Hi-C data. Additionally, it spatial distance matrices 2-Mb genomic regions provided FISH experiments. Furthermore, our suggests utilizing alone input sufficient predict emergence disappearance key TADs during early T cell differentiation. show in confined space can replicate both prevalence domain structures cell-to-cell variation positions observed single-cell lower preferentially form boundaries, this preference drives ensemble-averaged maps. The enrichment at CTCF binding sites be attributed influence on local model. Collectively, findings establish strong link between density, providing insights mechanisms underlying formation.

Language: Английский

Citations

0
Quantitative spatial analysis of chromatin biomolecular condensates using cryoelectron tomography DOI Creative Commons
Huabin Zhou, Joshua Hutchings, Momoko Shiozaki

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(19)

Published: May 6, 2025

Phase separation is an important mechanism to generate certain biomolecular condensates and organize the cell interior. Condensate formation function remain incompletely understood due difficulties in visualizing condensate interior at high resolution. Here, we analyzed structure of biochemically reconstituted chromatin through cryoelectron tomography. We found that traditional blotting methods sample preparation were inadequate, high-pressure freezing plus focused ion beam milling was essential maintain integrity. To identify densely packed molecules within condensate, integrated deep learning–based segmentation with context-aware template matching. Our approaches developed on also effective condensed regions situ native chromatin. Using these methods, determined average nucleosomes 6.1 12 Å resolution systems, respectively, form heterogeneous interaction networks both cases, gained insight into molecular origins surface tension condensates. should be applicable containing large distinctive components biochemical reconstitutions cellular systems.

Language: Английский

Citations

0
Location of oncogene-induced DNA damage sites revealed by quantitative analysis of a DNA counterstain DOI Creative Commons
Greta Paternò, Silvia Scalisi, Gaetano Ivan Dellino

et al.

European Biophysics Journal, Journal Year: 2025, Volume and Issue: unknown

Published: May 7, 2025

Language: Английский

Citations

0
The genome in space and time comes of age DOI Creative Commons
Martin Rey‐Millet, Kerstin Bystricky

Nucleus, Journal Year: 2024, Volume and Issue: 15(1)

Published: Feb. 1, 2024

DNA sequencing is not enough to grasp the complexity of genome organization and function. The four-dimensional (three in space, one time) configuration eukaryotic nucleus varies with cell types, during development diseased tissues, has be taken into account decipher To study, discuss, advance such direction, International Nucleome Consortium COST Action, funded by European Union, held its concluding symposium 'The Genome Space Time' at Ionian University Corfu, Greece, on September 10-13, 2023.

Language: Английский

Citations

2
Modulated illumination microscopy: Application perspectives in nuclear nanostructure analysis DOI Creative Commons
Christoph Cremer, Florian Schock, Antonio Virgilio Failla

et al.

Journal of Microscopy, Journal Year: 2024, Volume and Issue: 296(2), P. 121 - 128

Published: April 15, 2024

The structure of the cell nucleus higher organisms has become a major topic advanced light microscopy. So far, variety methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well different types patterned illumination modulated either laterally (in object plane) or axially (along optical axis). Based on our experience, we discuss here some application perspectives Modulated Illumination Microscopy (MIM) its combination with single-molecule (SMLM). For example, spatially microscopy/SMI (illumination modulation along axis) used to determine axial extension (size) small, optically isolated fluorescent objects between ≤ 200 nm ≥ 40 diameter precision down few range; it also allows positioning such structures 1 scale; combined structured illumination/SIM, 3D ≤1 is expected using yields typical for SMLM applications. Together nanosizing capability SMI, this can be analyse macromolecular nuclear complexes resolution approaching that cryoelectron

Language: Английский

Citations

2
Replication-dependent histone (Repli-Histo) labeling dissects the physical properties of euchromatin/heterochromatin in living human cells. DOI Creative Commons
Katsuhiko Minami, Satoru Ide, Kako Nakazato

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 21, 2024

Abstract A string of nucleosomes, where genomic DNA is wrapped around histones, organized in the cell as chromatin. Chromatin varies greatly, from euchromatin to heterochromatin, its genome functions. It important understand how heterochromatin physically different euchromatin. However, their specific labeling methods living cells are limited. To address this, we have developed replication-dependent histone (Repli-Histo labeling) label nucleosomes and based on replication timing. We investigated local nucleosome motion four chromatin classes human mouse cells. found that more euchromatic regions (earlier replicated regions) show greater motion. Notably, profile each class persists throughout interphase. Genome essentially with motions, even though timing program perturbed. Our findings, combined computational modeling, suggest earlier accessibility can be a major determinant genome-wide

Language: Английский

Citations

2
Approaches to characterize chromatin subcompartment organization in the cell nucleus DOI Open Access
Robin Weinmann, Lukas Frank, Karsten Rippe

et al.

Current Opinion in Structural Biology, Journal Year: 2023, Volume and Issue: 83, P. 102695 - 102695

Published: Sept. 16, 2023

Language: Английский

Citations

3
Transcription regulates the spatio-temporal dynamics of genes through micro-compartmentalization DOI Creative Commons

Hossein Salari,

Geneviève Fourel,

Daniel Jost

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: July 19, 2023

Abstract Although our understanding of the involvement heterochromatin architectural factors in shaping nuclear organization is improving, there still ongoing debate regarding role active genes this process. In study, we utilize publicly-available Micro-C data from mouse embryonic stem cells to investigate relationship between gene transcription and 3D folding. Our analysis uncovers a nonmonotonic - globally positive correlation intragenic contact density Pol II occupancy, independent cohesin-based loop extrusion. Through development biophysical model integrating dynamics within polymer chromosome organization, demonstrate that II-mediated attractive interactions with limited valency transcribed regions yield quantitative predictions consistent chromosome-conformation-capture live-imaging experiments. work provides compelling evidence transcriptional activity shapes 4D genome through micro-compartmentalization.

Language: Английский

Citations

2