scholarly journals Translation Microscopy (TRAM) for super-resolution imaging

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhen Qiu ◽  
Rhodri S Wilson ◽  
Yuewei Liu ◽  
Alison R Dun ◽  
Rebecca S Saleeb ◽  
...  
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Ground Truth ◽  
Image Plane ◽  
Ease Of Use ◽  
Stimulated Emission ◽  
Multiple Diffraction ◽  
Microscopy Technique ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Abstract Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

scholarly journals Super-Resolution Imaging With Lanthanide Luminescent Nanocrystals: Progress and Prospect

2021 ◽  
Vol 9 ◽  
Author(s):  
Hongxin Zhang ◽  
Mengyao Zhao ◽  
István M. Ábrahám ◽  
Fan Zhang
Keyword(s):  
Cell Biology ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Optical Resolution ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Lanthanide Ions ◽  
Fluorescence Probes ◽  
Saturation Intensity ◽  
Resolution Imaging

Stimulated emission depletion (STED) nanoscopy has overcome a serious diffraction barrier on the optical resolution and facilitated new discoveries on detailed nanostructures in cell biology. Traditional fluorescence probes employed in the super-resolution imaging approach include organic dyes and fluorescent proteins. However, some limitations of these probes, such as photobleaching, short emission wavelengths, and high saturation intensity, still hamper the promotion of optical resolution and bio-applications. Recently, lanthanide luminescent probes with unique optical properties of non-photobleaching and sharp emissions have been applied in super-resolution imaging. In this mini-review, we will introduce several different mechanisms for lanthanide ions to achieve super-resolution imaging based on an STED-like setup. Then, several lanthanide ions used in super-resolution imaging will be described in detail and discussed. Last but not least, we will emphasize the future challenges and outlooks in hope of advancing the next-generation lanthanide fluorescent probes for super-resolution optical imaging.

scholarly journals Lanthanide-Doped Upconversion Nanoparticles for Super-Resolution Microscopy

2021 ◽  
Vol 8 ◽  
Author(s):  
Hao Dong ◽  
Ling-Dong Sun ◽  
Chun-Hua Yan
Keyword(s):  
Cell Biology ◽  
Organic Dyes ◽  
Energy Levels ◽  
Super Resolution ◽  
Upconversion Nanoparticles ◽  
Non Invasive ◽  
Optimal Resolution ◽  
Resolution Imaging ◽  
Anti Stokes ◽  
Super Resolution Microscopy

Super-resolution microscopy offers a non-invasive and real-time tool for probing the subcellular structures and activities on nanometer precision. Exploring adequate luminescent probes is a great concern for acquiring higher-resolution image. Benefiting from the atomic-like transitions among real energy levels, lanthanide-doped upconversion nanoparticles are featured by unique optical properties including excellent photostability, large anti-Stokes shifts, multicolor narrowband emissions, tunable emission lifetimes, etc. The past few years have witnessed the development of upconversion nanoparticles as probes for super-resolution imaging studies. To date, the optimal resolution reached 28 nm (λ/36) for single nanoparticles and 82 nm (λ/12) for cytoskeleton structures with upconversion nanoparticles. Compared with conventional probes such as organic dyes and quantum dots, upconversion nanoparticle-related super-resolution microscopy is still in the preliminary stage, and both opportunities and challenges exist. In this perspective article, we summarized the recent advances of upconversion nanoparticles for super-resolution microscopy and projected the future directions of this emerging field. This perspective article should be enlightening for designing efficient upconversion nanoprobes for super-resolution imaging and promote the development of upconversion nanoprobes for cell biology applications.

Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach

Nanoscale ◽  
2018 ◽  
Vol 10 (34) ◽  
pp. 16252-16260 ◽  
Author(s):  
Luwei Wang ◽  
Bingling Chen ◽  
Wei Yan ◽  
Zhigang Yang ◽  
Xiao Peng ◽  
...  
Keyword(s):  
Low Power ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Resolution Limit ◽  
Sted Microscopy ◽  
Microscopy Technique ◽  
Resolution Improvement ◽  
Phasor Plot ◽  
Stimulated Emission Depletion ◽  
Super Resolution Microscopy

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that has achieved significant results in breaking the resolution limit and relevant applications.

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.

scholarly journals MINSTED fluorescence localization and nanoscopy

2021 ◽  
Author(s):  
Michael Weber ◽  
Marcel Leutenegger ◽  
Stefan Stoldt ◽  
Stefan Jakobs ◽  
Tiberiu S. Mihaila ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stimulated Emission ◽  
Far Field ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Molecular Scale ◽  
Localization Precision ◽  
Super Resolution Microscopy

AbstractWe introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

scholarly journals Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246138
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

Recent advances in luminescent materials for super-resolution imaging via stimulated emission depletion nanoscopy

2021 ◽  
Author(s):  
Yanzi Xu ◽  
Ruohan Xu ◽  
Zhi Wang ◽  
Yu Zhou ◽  
Qifei Shen ◽  
...  
Keyword(s):  
Recent Progress ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Luminescent Materials ◽  
New Materials ◽  
Recent Advances ◽  
Resolution Imaging ◽  
Stimulated Emission Depletion

Recent progress on STED fluorophores for super-resolution imaging and also their characteristics are outlined here, thus providing some guidelines to select proper probes and even develop new materials for super-resolution imaging via STED nanoscopy.

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