scholarly journals Three-dimensional super-resolution fluorescence imaging of DNA

2019 ◽  
Author(s):  
Sevim Yardimci ◽  
Daniel R. Burnham ◽  
Samantha Y. A. Terry ◽  
Hasan Yardimci
Keyword(s):  
Three Dimensional ◽  
Super Resolution ◽  
Structural Features ◽  
Cellular Structures ◽  
Complex Environment ◽  
Metaphase Chromosomes ◽  
Single Dna Molecules ◽  
Dna Intercalators ◽  
Double Stranded Dna ◽  
Super Resolution Microscopy

AbstractRecent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of mono-over bis-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.

scholarly journals Isotropic Three-Dimensional Dual-Color Super-Resolution Microscopy with Metal-Induced Energy Transfer

2021 ◽  
Author(s):  
Jan Christoph Thiele ◽  
Marvin Jungblut ◽  
Dominic A. Helmerich ◽  
Roman Tsukanov ◽  
Anna Chizhik ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Three Dimensions ◽  
Lateral Resolution ◽  
Cellular Structures ◽  
Axial Resolution ◽  
Dual Color ◽  
Super Resolution Microscopy

Over the last two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution. Similar to conventional optical microscopy, the axial resolution is by a factor three to five worse than the lateral resolution. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging which achieves an axial resolution down to nanometers. It exploits the distance dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of sub-cellular structures. Moreover, we employed spectral demixing for implementing dual-color MIET-dSTORM that allows us to image and co-localize, in three dimensions, two different cellular structures simultaneously.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

Cubic spline-based depth-dependent localization of mitochondria–endoplasmic reticulum contacts by three-dimensional light-sheet super-resolution microscopy

The Analyst ◽  
2021 ◽  
Author(s):  
Yucheng Sun ◽  
Seungah Lee ◽  
Seong Ho Kang
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Homeostasis ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Cubic Spline ◽  
Contact Distance ◽  
Super Resolution Microscopy

The contact distance between mitochondria (Mito) and endoplasmic reticulum (ER) has received considerable attention owing to their crucial function in maintaining lipid and calcium homeostasis. Herein, cubic spline algorithm-based depth-dependent...

scholarly journals Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

2014 ◽  
Vol 136 (40) ◽  
pp. 14003-14006 ◽  
Author(s):  
Marissa K. Lee ◽  
Prabin Rai ◽  
Jarrod Williams ◽  
Robert J. Twieg ◽  
W. E. Moerner
Keyword(s):  
Small Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cell ◽  
Cell Surfaces ◽  
Super Resolution Microscopy

scholarly journals A bisected pupil for studying single-molecule orientational dynamics and its application to three-dimensional super-resolution microscopy

2014 ◽  
Vol 104 (19) ◽  
pp. 193701 ◽  
Author(s):  
Adam S. Backer ◽  
Mikael P. Backlund ◽  
Alexander R. von Diezmann ◽  
Steffen J. Sahl ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Orientational Dynamics ◽  
Super Resolution Microscopy
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