scholarly journals Deformed Alignment of Super-Resolution Images for Semi-flexible Structures in 3D

2018 ◽  
Author(s):  
Xiaoyu Shi ◽  
Galo Garcia ◽  
Yina Wang ◽  
Jeremy Reiter ◽  
Bo Huang
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Flexible Structures ◽  
Super Resolution ◽  
Computation Time ◽  
Structural Heterogeneity ◽  
Image Alignment ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
Super Resolution Microscopy

AbstractDue to low labeling efficiency and structural heterogeneity in fluorescence-based single-molecule localization microscopy (SMLM), image alignment and quantitative analysis is often required to make accurate conclusions on the spatial relationships between proteins. Cryo-electron microscopy (EM) image alignment procedures have been applied to average structures taken with super-resolution microscopy. However, unlike cryo-EM, the much larger cellular structures analyzed by super-resolution microscopy are often heterogeneous, resulting in misalignment. And the light-microscopy image library is much smaller, which makes classification not realistic. To overcome these two challenges, we developed a method to deform semi-flexible ring-shaped structures and then align the 3D structures without classification. These algorithms can register semi-flexible structures with an accuracy of several nanometers in short computation time and with greatly reduced memory requirements. We demonstrated our methods by aligning experimental Stochastic Optical Reconstruction Microscopy (STORM) images of ciliary distal appendages and simulated structures. Symmetries, dimensions, and locations of protein complexes in 3D are revealed by the alignment and averaging for heterogeneous, tilted, and under-labeled structures.

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.

Optical super-resolution microscopy unravels the molecular composition of functional protein complexes

Nanoscale ◽  
2019 ◽  
Vol 11 (39) ◽  
pp. 17981-17991 ◽  
Author(s):  
Marina S. Dietz ◽  
Mike Heilemann
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Super Resolution ◽  
Molecular Composition ◽  
Functional Protein ◽  
Super Resolution Microscopy

The molecular composition of functional protein complexes can be determined from single-molecule super-resolution images.

scholarly journals Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity

2020 ◽  
Author(s):  
Paramita Ray ◽  
Krishnan Raghunathan ◽  
Aarif Ahsan ◽  
Uday Sankar Allam ◽  
Shirish Shukla ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Super Resolution ◽  
Receptor Internalization ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Steady State Levels ◽  
Epidermal Growth ◽  
Optical Reconstruction ◽  
Super Resolution Microscopy

ABSTRACTWe previously reported that differential protein degradation of TKI-sensitive [L858R, del(E746-A750)] and resistant (T790M) epidermal growth factor receptor (EGFR) mutants upon erlotinib treatment correlates with drug sensitivity. However, the molecular mechanism remains unclear. We also reported SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is critical in receptor stability and TKI sensitivity. We found that L858R/T790M EGFR is a preferred substrate of SMURF2-UBCH5 (an E3-E2) complex-mediated K63-linked polyubiquitination, which preferentially stabilizes mutant receptor. We identified four lysine (K) residues (K721, 846, 1037 and 1164) as the sites of ubiquitination and replacement of K to acetylation-mimicking asparagine (Q) at K1037 position in L858R/T790M background converts the stable protein sensitive to erlotinib-induced degradation. Using STochastic Optical Reconstruction Microscopy (STORM) imaging, we show that SMURF2 presence allows longer membrane retention of activated EGFR upon EGF treatment, whereas, siRNA-mediated SMURF2 knockdown fastens receptor endocytosis and lysosome enrichment. In an erlotinib-sensitive PC9 cells, SMURF2 overexpression increased EGFR levels with improved erlotinib tolerance, whereas, SMURF2 knockdown decreased EGFR steady state levels in NCI-H1975 and PC9-AR cells to overcome erlotinib and AZD-9291 resistance respectively. Additionally, by genetically altering the SMURF2-UBCH5 complex formation destabilized EGFR. Together, we propose that SMURF2-mediated preferential polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization to provide enhanced receptor stability and that disruption of the E3-E2 complex may be an attractive alternate to overcome TKI resistance.

scholarly journals Vectashield-induced fluorescence quenching hinders conventional and super resolution microscopy

2018 ◽  
Author(s):  
Aleksandra Arsić ◽  
Nevena Stajković ◽  
Rainer Spiegel ◽  
Ivana Nikić-Spiegel
Keyword(s):  
Fluorescence Intensity ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Practical Advice ◽  
Alexa Fluor ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Conventional Microscopy ◽  
Optical Reconstruction ◽  
The Right ◽  
Super Resolution Microscopy

AbstractFinding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent ON state. This helps identifying structures of interests. Subsequently, labelled samples are brought to blinking, which is necessary for localization of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor (AF) 647, blinks in Vectashield. However, after adding Vectashield to our samples, we noticed that the fluorescence intensity of AF647 and its improved variant, AF647+, is quenched. Since structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) practical advice on how to use Vectashield for dSTORM imaging of AF647 and AF647+.

scholarly journals Tetra-gel enables superior accuracy in combined super-resolution imaging and expansion microscopy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hsuan Lee ◽  
Chih-Chieh Yu ◽  
Edward S. Boyden ◽  
Xiaowei Zhuang ◽  
Pallav Kosuri
Keyword(s):  
Standard Deviation ◽  
Single Molecule ◽  
Super Resolution ◽  
Structural Features ◽  
Chain Growth ◽  
Radical Chain ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Direct Measurements ◽  
Optical Reconstruction ◽  
Structural Preservation

AbstractThe accuracy of expansion microscopy (ExM) depends on the structural preservation of samples embedded in a hydrogel. However, it has been unknown to what extent gel embedding alters the molecular positions of individual labeled sites. Here, we quantified the accuracy of gel embedding by using stochastic optical reconstruction microscopy (STORM) to image DNA origami with well-defined structures. We found that embedding in hydrogels based on polyacrylamide, the most widely used chemistry in ExM, resulted in random displacements of labeled sites with a standard deviation of ~ 16 nm. In contrast, we found that embedding in tetra-gel, a hydrogel that does not depend on free-radical chain-growth polymerization, preserved labeled sites with a standard deviation of less than 5 nm. By combining tetra-gel ExM with STORM, we were able to resolve 11-nm structural features without the loss in accuracy seen with polyacrylamide gels. Our study thus provides direct measurements of the single-molecule distortions resulting from hydrogel embedding, and presents a way to improve super-resolution microscopy through combination with tetra-gel ExM.

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.

scholarly journals Volumetric super-resolution imaging by serial ultrasectioning and STochastic Optical Reconstruction Microscopy (STORM) in neural tissue

2021 ◽  
Author(s):  
Tarlan Vatan ◽  
Jacqueline A. Minehart ◽  
Chenghang Zhang ◽  
Vatsal Agarwal ◽  
Jerry Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Neural Tissue ◽  
Imaging Data ◽  
Whole Cells ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Tissue Scattering ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Chemical Synapses

Abstract Here we present a protocol for collecting large-volume, four-color, single-molecule localization imaging data from neural tissue. We have applied this technique to map the location and identities of chemical synapses across whole cells in mouse retinae. Our sample preparation approach improves 3D STORM image quality by reducing tissue scattering, photobleaching, and optical distortions associated with deep imaging. This approach can be extended for use on other tissue types enabling life scientists to perform volumetric super-resolution imaging in diverse biological models. For a detailed application of this protocol, please refer to Sigal et al., 2015.

scholarly journals Toward Sub-Diffraction Imaging of Single-DNA Molecule Sensors Based on Stochastic Switching Localization Microscopy

Sensors ◽  
2020 ◽  
Vol 20 (22) ◽  
pp. 6667
Author(s):  
Seungah Lee ◽  
Indra Batjikh ◽  
Seong Ho Kang
Keyword(s):  
Single Molecule ◽  
Specific Binding ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Single Molecule Level ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Wide Range ◽  
Nanoscale Topography ◽  
Diffraction Imaging ◽  
Optical Reconstruction

The natural characteristics of deoxyribonucleic acid (DNA) enable its advanced applications in nanotechnology as a special tool that can be detected by high-resolution imaging with precise localization. Super-resolution (SR) microscopy enables the examination of nanoscale molecules beyond the diffraction limit. With the development of SR microscopy methods, DNA nanostructures can now be optically assessed. Using the specific binding of fluorophores with their target molecules, advanced single-molecule localization microscopy (SMLM) has been expanded into different fields, allowing wide-range detection at the single-molecule level. This review discusses the recent progress in the SR imaging of DNA nano-objects using SMLM techniques, such as direct stochastic optical reconstruction microscopy, binding-activated localization microscopy, and point accumulation for imaging nanoscale topography. Furthermore, we discuss their advantages and limitations, present applications, and future perspectives.

scholarly journals Self-activated photoblinking of nitrogen vacancy centers in nanodiamonds (sandSTORM): A method for rapid single molecule localization microscopy with unlimited observation time

2020 ◽  
Author(s):  
Kaarjel K. Narayanasamy ◽  
Joshua C. Price ◽  
Raquel Mesquita-Riberio ◽  
Melissa L. Mather ◽  
Izzy Jayasinghe
Keyword(s):  
Single Molecule ◽  
Light Exposure ◽  
Super Resolution ◽  
Observation Time ◽  
Nitrogen Vacancy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Nitrogen Vacancy Centers ◽  
Optical Reconstruction ◽  
Living Neurons ◽  
Microscopy Techniques

AbstractStochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the ‘off’-rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV0and NV- is localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at ∼ 3-times the speed and ∼ 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.

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.

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