Pushing the super-resolution limit: recent improvements in microscopy below the diffraction limit

2021 ◽  
Author(s):  
D. J. Nieves ◽  
M. A. B. Baker
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Information ◽  
Biological Systems ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Resolving Power ◽  
Resolution Limit ◽  
Single Protein ◽  
Super Resolution Microscopy ◽  
Near Future

Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the ‘super-resolution’ limit and what opportunities remain for further improvements in the near future.

scholarly journals The Nanoscopic Organization of Synapse Structures: A Common Basis for Cell Communication

Membranes ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 248
Author(s):  
Xiaojuan Yang ◽  
Wim Annaert
Keyword(s):  
Plasma Membranes ◽  
Current Knowledge ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stimulated Emission ◽  
Synaptic Proteins ◽  
Resolving Power ◽  
Structured Illumination Microscopy ◽  
Membrane Contact Sites ◽  
Super Resolution Microscopy

Synapse structures, including neuronal and immunological synapses, can be seen as the plasma membrane contact sites between two individual cells where information is transmitted from one cell to the other. The distance between the two plasma membranes is only a few tens of nanometers, but these areas are densely populated with functionally different proteins, including adhesion proteins, receptors, and transporters. The narrow space between the two plasma membranes has been a barrier for resolving the synaptic architecture due to the diffraction limit in conventional microscopy (~250 nm). Various advanced super-resolution microscopy techniques, such as stimulated emission depletion (STED), structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM), bypass the diffraction limit and provide a sub-diffraction-limit resolving power, ranging from 10 to 100 nm. The studies using super-resolution microscopy have revealed unprecedented details of the nanoscopic organization and dynamics of synaptic molecules. In general, most synaptic proteins appear to be heterogeneously distributed and form nanodomains at the membranes. These nanodomains are dynamic functional units, playing important roles in mediating signal transmission through synapses. Herein, we discuss our current knowledge on the super-resolution nanoscopic architecture of synapses and their functional implications, with a particular focus on the neuronal synapses and immune synapses.

scholarly journals Protein-, Polymer-, and Silica-Based Luminescent Nanomaterial Probes for Super Resolution Microscopy: A Review

2021 ◽  
Author(s):  
Shelby B Thompson ◽  
Dimitri Pappas
Keyword(s):  
Optical Microscopy ◽  
Biological Systems ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Protein Polymer ◽  
Super Resolution Microscopy

Super resolution microscopy was developed to overcome the Abbe Diffraction Limit, which effects conventional optical microscopy in order to study the smaller components of biological systems. In recent years nanomaterials...

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.

Super-resolution FRET measurements

Nanoscale ◽  
2021 ◽  
Author(s):  
Fernando D Stefani ◽  
Alan M. M. Szalai ◽  
Cecilia Zaza
Keyword(s):  
Energy Transfer ◽  
Fluorescence Microscopy ◽  
Biological Systems ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Dynamic Architecture ◽  
Förster Resonance

Super-resolution fluorescence microscopy and Förster Resonance Energy Transfer (FRET) form a well-established family of techniques that has provided unique tools to study the dynamic architecture and functionality of biological systems,...

An overview of structured illumination microscopy: recent advances and perspectives

2021 ◽  
Author(s):  
Krishnendu Samanta ◽  
Joby Joseph
Keyword(s):  
Spatial Frequency ◽  
Basic Principle ◽  
Imaging Techniques ◽  
Resolution Enhancement ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Structured Illumination ◽  
Post Processing ◽  
Structured Illumination Microscopy ◽  
Near Future

Abstract Structured illumination microscopy (SIM) is one of the most significant widefield super-resolution optical imaging techniques. The conventional SIM utilizes a sinusoidal structured pattern to excite the fluorescent sample; which eventually down-modulates higher spatial frequency sample information within the diffraction-limited passband of the microscopy system and provides around two-fold resolution enhancement over diffraction limit after suitable computational post-processing. Here we provide an overview of the basic principle, image reconstruction, technical development of the SIM technique. Nonetheless, in order to push the SIM resolution further towards the extreme nanoscale dimensions, several different approaches are launched apart from the conventional SIM. Among the various SIM methods, some of the important techniques e.g. TIRF, non-linear, plasmonic, speckle SIM etc. are discussed elaborately. Moreover, we highlight different implementations of SIM in various other imaging modalities to enhance their imaging performances with augmented capabilities. Finally, some future outlooks are mentioned which might develop fruitfully and pave the way for new discoveries in near future.

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.

Two-Point Separation in Far-Field Super-Resolution Fluorescence Microscopy Based on Two-Color Fluorescence Dip Spectroscopy, Part I: Experimental Evaluation

2005 ◽  
Vol 59 (7) ◽  
pp. 868-872 ◽  
Author(s):  
Takeshi Watanabe ◽  
Yoshinori Iketaki ◽  
Takashige Omatsu ◽  
Kimihisa Yamamoto ◽  
Masaaki Fujii
Keyword(s):  
Fluorescence Microscopy ◽  
Phase Modulation ◽  
Laser Light ◽  
Super Resolution ◽  
Separation Distance ◽  
Diffraction Limit ◽  
Far Field ◽  
Fluorescent Beads ◽  
Fluorescent Image ◽  
Point Separation

The two-point resolution of a novel two-color far-field super-resolution fluorescence microscopy was evaluated by measuring fluorescent beads 100 nm in diameter. This microscopy is based on a combination of two-color fluorescence dip spectroscopy and a phase-modulation technique for a laser beam. By simply introducing two-color laser light, the size of the fluorescent image of a bead was shrunk down to a diameter of 250 nm from the diffraction-limited image with a diameter of 360 nm. For two closely adjacent fluorescent beads with a separation distance of 350 nm, the two-color microscope clearly gave separated fluorescence images, while the conventional one-color fluorescence microscope could not resolve them. It has been proved that our technique breaks Rayleigh's diffraction limit.

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 microscopy and its applications in neuroscience

2017 ◽  
Vol 10 (05) ◽  
pp. 1730001 ◽  
Author(s):  
Xuecen Wang ◽  
Jiahao Wang ◽  
Xinpei Zhu ◽  
Yao Zheng ◽  
Ke Si ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Biological Science ◽  
Basic Principles ◽  
Suitable Tool ◽  
Subcellular Processes ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

Optical microscopy promises researchers to see most tiny substances directly. However, the resolution of conventional microscopy is restricted by the diffraction limit. This makes it a challenge to observe subcellular processes happened in nanoscale. The development of super-resolution microscopy provides a solution to this challenge. Here, we briefly review several commonly used super-resolution techniques, explicating their basic principles and applications in biological science, especially in neuroscience. In addition, characteristics and limitations of each technique are compared to provide a guidance for biologists to choose the most suitable tool.

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