scholarly journals In Situ Imaging of Spatial Organization of Accessible Chromatin at the Nanoscale with ATAC-see and Single-Molecule Super-Resolution Fluorescence Microscopy

2018 ◽  
Vol 114 (3) ◽  
pp. 539a
Author(s):  
Maurice Y. Lee ◽  
Xingqi Chen ◽  
Anna-Karin Gustavsson ◽  
Howard Y. Chang ◽  
W.E. Moerner
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Accessible Chromatin ◽  
In Situ Imaging

scholarly journals A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair

2013 ◽  
Vol 202 (3) ◽  
pp. 579-595 ◽  
Author(s):  
Sébastien Britton ◽  
Julia Coates ◽  
Stephen P. Jackson
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
Anticancer Agents ◽  
Spatial Organization ◽  
System Development ◽  
Super Resolution ◽  
Strand Break ◽  
Resistance Mechanisms ◽  
Original Method ◽  
Immune System Development

DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair factors at DSBs in cells. In this paper, we present an original method that involves a combination of ribonuclease- and detergent-based preextraction with high-resolution microscopy. This method allows direct visualization of previously hidden repair complexes, including the main DSB sensor Ku, at virtually any type of DSB, including those induced by anticancer agents. We demonstrate its broad range of applications by coupling it to laser microirradiation, super-resolution microscopy, and single-molecule counting to investigate the spatial organization and composition of repair factories. Furthermore, we use our method to monitor DNA repair and identify mechanisms of repair pathway choice, and we show its utility in defining cellular sensitivities and resistance mechanisms to anticancer agents.

scholarly journals Single Molecule Localization Microscopy (SMLM) imaging of bacteria: A sample preparation guide

2020 ◽  
Author(s):  
Sachith D. Gunasinghe ◽  
Kirstin D. Elgass ◽  
Toby D. M. Bell ◽  
Trevor Lithgow
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Single Molecule ◽  
Spatial Organization ◽  
Outer Membrane Proteins ◽  
Fluorescent Microscopy ◽  
Super Resolution ◽  
Model Systems ◽  
Invaluable Tool ◽  
Optical Reconstruction

Abstract In recent years Super-resolution microscopy has become an invaluable tool to noninvasively interrogate the membrane architecture of bacteria to study the spatial organization of proteins associated with membranes, which in turn help us to understand how bacteria have evolved to exploit environmental niches. Model systems like Escherichia coli and Caulobacter cresentus have been used to study the spatiotemporal organization of membrane proteins. Like most gram-negative bacteria, the outer membrane of E.coli is populated with β-barrel proteins, which serve as selective channels where exchange of small molecules take place. Surface exposed domains in these channels provide means to fluorescently label and utilise them for fluorescent microscopy studies to investigate their spatial organization at the outer membrane. Here, we describe a methodology to fluorescently label outer membrane proteins in E.coli and study their spatial organization using direct stochastic optical reconstruction microscopy (dSTORM).

scholarly journals Spatial Organization of Transcription in E. Coli using Super-Resolution Fluorescence Microscopy

2017 ◽  
Vol 112 (3) ◽  
pp. 212a
Author(s):  
Xiaoli Weng ◽  
Christopher Bohrer ◽  
Arvin Lagda ◽  
Jie Xiao
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Organization ◽  
Super Resolution ◽  
E Coli

scholarly journals Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy

2020 ◽  
Vol 6 (22) ◽  
pp. eaba4542 ◽  
Author(s):  
Chenyi Mao ◽  
Min Yen Lee ◽  
Jing-Ru Jhan ◽  
Aaron R. Halpern ◽  
Marcus A. Woodworth ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Processing Conditions ◽  
Sample Processing ◽  
Tissue Fluorescence ◽  
Uniform Labeling ◽  
Tissue Microscopy

Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.

scholarly journals In Situ Visualization of Block Copolymer Self-Assembly in Organic Media by Super-Resolution Fluorescence Microscopy

2015 ◽  
Vol 21 (51) ◽  
pp. 18539-18542 ◽  
Author(s):  
Charlotte E. Boott ◽  
Romain F. Laine ◽  
Pierre Mahou ◽  
John R. Finnegan ◽  
Erin M. Leitao ◽  
...  
Keyword(s):  
Block Copolymer ◽  
Fluorescence Microscopy ◽  
Self Assembly ◽  
Super Resolution ◽  
Organic Media ◽  
In Situ Visualization

scholarly journals Super-resolution Localization and Defocused Fluorescence Microscopy on Resonantly Coupled Single-Molecule, Single-Nanorod Hybrids

ACS Nano ◽  
2016 ◽  
Vol 10 (2) ◽  
pp. 2455-2466 ◽  
Author(s):  
Liang Su ◽  
Haifeng Yuan ◽  
Gang Lu ◽  
Susana Rocha ◽  
Michel Orrit ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Super Resolution

scholarly journals Characterizing Locus Specific Chromatin Structure and Dynamics with Correlative Conventional and Super Resolution imaging in living cells

2021 ◽  
Author(s):  
Dushyant Mehra ◽  
Santosh Adhikari ◽  
Chiranjib Banerjee ◽  
Elias M. Puchner
Keyword(s):  
Single Molecule ◽  
Chromatin Structure ◽  
Spatial Organization ◽  
Super Resolution ◽  
Living Cells ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Acquisition Time ◽  
Structure And Dynamics ◽  
Chromatin Structure And Dynamics

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution PALM imaging precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and searching dCas9 molecules, whose mobility overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule precision and yields unprecedented insights across length and time scales.

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