Super-­resolution imaging with stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM)

2014 ◽  
pp. 61-84
Author(s):  
Yujie Sun
Keyword(s):  
Super Resolution ◽  
Localization Microscopy ◽  
Photoactivated Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction

Biological Insight from Super-Resolution Microscopy: What We Can Learn from Localization-Based Images

2018 ◽  
Vol 87 (1) ◽  
pp. 965-989 ◽  
Author(s):  
David Baddeley ◽  
Joerg Bewersdorf
Keyword(s):  
Imaging Modality ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Photoactivated Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Biological Insight ◽  
Biological Discovery ◽  
Optical Reconstruction ◽  
Super Resolution Microscopy ◽  
Fluorescent Molecules

Super-resolution optical imaging based on the switching and localization of individual fluorescent molecules [photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), etc.] has evolved remarkably over the last decade. Originally driven by pushing technological limits, it has become a tool of biological discovery. The initial demand for impressive pictures showing well-studied biological structures has been replaced by a need for quantitative, reliable data providing dependable evidence for specific unresolved biological hypotheses. In this review, we highlight applications that showcase this development, identify the features that led to their success, and discuss remaining challenges and difficulties. In this context, we consider the complex topic of defining resolution for this imaging modality and address some of the more common analytical methods used with this data.

scholarly journals Single-molecule Photoswitching and Localization

2011 ◽  
Vol 64 (5) ◽  
pp. 503 ◽  
Author(s):  
Sebastian van de Linde ◽  
Steve Wolter ◽  
Markus Sauer
Keyword(s):  
Single Molecule ◽  
Optical Resolution ◽  
Super Resolution ◽  
Image Plane ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Optical Reconstruction ◽  
20 Nm

Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ∼20 nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.

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.

Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging

Nanoscale ◽  
2015 ◽  
Vol 7 (6) ◽  
pp. 2511-2519 ◽  
Author(s):  
Jing Gao ◽  
Ye Wang ◽  
Mingjun Cai ◽  
Yangang Pan ◽  
Haijiao Xu ◽  
...  
Keyword(s):  
Distribution Pattern ◽  
Lipid Rafts ◽  
Essential Role ◽  
Super Resolution ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Polarized Cells ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Cluster Maintenance

We investigate the distribution of membrane EGFR by direct stochastic optical reconstruction microscopy (dSTORM). Our results illustrate the clustering distribution pattern of EGFR in polarized cells and uncover the essential role of lipid rafts in EGFR cluster maintenance.

scholarly journals Volumetric super-resolution imaging by serial ultrasectioning and stochastic optical reconstruction microscopy in mouse neural tissue

2021 ◽  
Vol 2 (4) ◽  
pp. 100971
Author(s):  
Tarlan Vatan ◽  
Jacqueline A. Minehart ◽  
Chenghang Zhang ◽  
Vatsal Agarwal ◽  
Jerry Yang ◽  
...  
Keyword(s):  
Super Resolution ◽  
Neural Tissue ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction

scholarly journals Surface Charge Manipulation and Electrostatic Immobilization of Synaptosomes for Super-resolution Imaging: A Study on Tau Compartmentalization

2021 ◽  
Author(s):  
Ushashi Bhattacharya ◽  
Jia-Fong Jhou ◽  
Yi-Fong Zou ◽  
Gerald Abrigo ◽  
Shu-Wei Lin ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Protein Localization ◽  
Super Resolution ◽  
Synaptic Terminals ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Neural Transmission ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Cortical Synapses ◽  
Induced Aggregation

Abstract Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a simple and facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.

scholarly journals About samples, giving examples: Optimized Single Molecule Localization Microscopy

2019 ◽  
Author(s):  
Angélique Jimenez ◽  
Karoline Friedl ◽  
Christophe Leterrier
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Full Potential ◽  
Coated Pits ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Nanoscale Topography ◽  
Biological Discovery ◽  
Optical Reconstruction ◽  
Single Molecule Localization Microscopy

AbstractSuper-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

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