scholarly journals Visualizing and discovering cellular structures with super-resolution microscopy

Science ◽  
2018 ◽  
Vol 361 (6405) ◽  
pp. 880-887 ◽  
Author(s):  
Yaron M. Sigal ◽  
Ruobo Zhou ◽  
Xiaowei Zhuang
Keyword(s):  
State Of The Art ◽  
Super Resolution ◽  
Building Blocks ◽  
Diffraction Limit ◽  
Three Dimensions ◽  
Cellular Structures ◽  
Nanometer Scale ◽  
Molecular Building Blocks ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Super-resolution microscopy has overcome a long-held resolution barrier—the diffraction limit—in light microscopy and enabled visualization of previously invisible molecular details in biological systems. Since their conception, super-resolution imaging methods have continually evolved and can now be used to image cellular structures in three dimensions, multiple colors, and living systems with nanometer-scale resolution. These methods have been applied to answer questions involving the organization, interaction, stoichiometry, and dynamics of individual molecular building blocks and their integration into functional machineries in cells and tissues. In this Review, we provide an overview of super-resolution methods, their state-of-the-art capabilities, and their constantly expanding applications to biology, with a focus on the latter. We will also describe the current technical challenges and future advances anticipated in super-resolution imaging.

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 Fluorophore-labelled RNA aptamers to common protein tags as super-resolution imaging reagents.

2020 ◽  
Author(s):  
Juan Wang ◽  
Avtar Singh ◽  
Abdullah Ozer ◽  
Warren R Zipfel
Keyword(s):  
Fluorescent Protein ◽  
Super Resolution ◽  
Rna Aptamers ◽  
Cellular Structures ◽  
Intracellular Proteins ◽  
Resolution Imaging ◽  
Green Fluorescent ◽  
Protein Tags ◽  
Super Resolution Microscopy ◽  
Conventional Antibody

Developing labelling methods that densely and specifically label targeted cellular structures is critically important for centroid localization-based super-resolution microscopy. Being easy and inexpensive to produce in the laboratory and of relatively small size, RNA aptamers have potential as a substitute for conventional antibody labelling. By using aptamers selected against common protein tags - GFP (green fluorescent protein) in this case - we demonstrate labelling methods using dSTORM-compatible fluorophores for STORM and hybridizable imager strands for DNA-PAINT super-resolution optical imaging of any cellular proteins fused to the aptamer binding target. We show that we can label both extracellular and intracellular proteins for super-resolution imaging, and that the method in particular, offers some interesting advantages for live cell super-resolution imaging of plasma membrane proteins.

scholarly journals Light Microscopy of Mitochondria at the Nanoscale

2020 ◽  
Vol 49 (1) ◽  
pp. 289-308 ◽  
Author(s):  
Stefan Jakobs ◽  
Till Stephan ◽  
Peter Ilgen ◽  
Christian Brüser
Keyword(s):  
Light Microscopy ◽  
Mitochondrial Function ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Cellular Structures ◽  
Inner Membrane ◽  
Double Membrane ◽  
Future Developments ◽  
Super Resolution Microscopy ◽  
Conventional Light Microscopy

Mitochondria are essential for eukaryotic life. These double-membrane organelles often form highly dynamic tubular networks interacting with many cellular structures. Their highly convoluted contiguous inner membrane compartmentalizes the organelle, which is crucial for mitochondrial function. Since the diameter of the mitochondrial tubules is generally close to the diffraction limit of light microscopy, it is often challenging, if not impossible, to visualize submitochondrial structures or protein distributions using conventional light microscopy. This renders super-resolution microscopy particularly valuable, and attractive, for studying mitochondria. Super-resolution microscopy encompasses a diverse set of approaches that extend resolution, as well as nanoscopy techniques that can even overcome the diffraction limit. In this review, we provide an overview of recent studies using super-resolution microscopy to investigate mitochondria, discuss the strengths and opportunities of the various methods in addressing specific questions in mitochondrial biology, and highlight potential future developments.

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 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.

Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes

Nano Letters ◽  
2015 ◽  
Vol 15 (2) ◽  
pp. 1374-1381 ◽  
Author(s):  
Simon Hennig ◽  
Sebastian van de Linde ◽  
Martina Lummer ◽  
Matthias Simonis ◽  
Thomas Huser ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Super Resolution ◽  
Live Cell ◽  
Cellular Structures ◽  
Resolution Imaging

scholarly journals Super-resolution microscopy: successful applications in centrosome study and beyond

2019 ◽  
Vol 5 (5-6) ◽  
pp. 235-243 ◽  
Author(s):  
Jingyan Fu ◽  
Chuanmao Zhang
Keyword(s):  
Molecular Basis ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
The Past ◽  
Eukaryotic Species ◽  
Organizing Center ◽  
Cilia And Flagella ◽  
Super Resolution Microscopy

AbstractCentrosome is the main microtubule-organizing center in most animal cells. Its core structure, centriole, also assembles cilia and flagella that have important sensing and motility functions. Centrosome has long been recognized as a highly conserved organelle in eukaryotic species. Through electron microscopy, its ultrastructure was revealed to contain a beautiful nine-symmetrical core 60 years ago, yet its molecular basis has only been unraveled in the past two decades. The emergence of super-resolution microscopy allows us to explore the insides of a centrosome, which is smaller than the diffraction limit of light. Super-resolution microscopy also enables the compartmentation of centrosome proteins into different zones and the identification of their molecular interactions and functions. This paper compiles the centrosome architecture knowledge that has been revealed in recent years and highlights the power of several super-resolution techniques.

Super-resolution imaging of SERS hot spots

2014 ◽  
Vol 43 (11) ◽  
pp. 3854-3864 ◽  
Author(s):  
Katherine A. Willets
Keyword(s):  
Hot Spots ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Resolution Imaging

Super-resolution imaging defeats the diffraction-limit of light, allowing the spatial origin and intensity of SERS signals to be determined with <5 nm resolution.

scholarly journals Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells

Nanoscale ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 10023-10033 ◽  
Author(s):  
Jan Bergstrand ◽  
Lei Xu ◽  
Xinyan Miao ◽  
Nailin Li ◽  
Ozan Öktem ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Dictionary Learning ◽  
Super Resolution ◽  
Distribution Patterns ◽  
Specific Protein ◽  
Protein Distribution ◽  
Activated Platelets ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Super-resolution imaging of P-selectin in platelets together with dictionary learning allow specifically activated platelets to be identified in an automatic objective manner.

scholarly journals Super-Resolution Microscopy of Chromatin

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 493 ◽  
Author(s):  
Birk
Keyword(s):  
Gene Regulation ◽  
Data Analysis ◽  
Super Resolution ◽  
Fluorescence Labeling ◽  
Cellular Processes ◽  
Direct Assessment ◽  
Chromatin Interactions ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Insight Into

Since the advent of super-resolution microscopy, countless approaches and studies have been published contributing significantly to our understanding of cellular processes. With the aid of chromatin-specific fluorescence labeling techniques, we are gaining increasing insight into gene regulation and chromatin organization. Combined with super-resolution imaging and data analysis, these labeling techniques enable direct assessment not only of chromatin interactions but also of the function of specific chromatin conformational states.

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