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+.

Sodium sulfite as a switching agent for single molecule based super-resolution optical microscopy

2021 ◽  
Author(s):  
Anders K Engdahl ◽  
Oleg Grauberger ◽  
Mark Schüttpelz ◽  
Thomas Huser
Keyword(s):  
Single Molecule ◽  
Fluorescent Dyes ◽  
Sodium Sulfite ◽  
Super Resolution ◽  
Oxygen Scavenger ◽  
Dark State ◽  
Alexa Fluor ◽  
High Illumination ◽  
Human Osteosarcoma Cells ◽  
Super Resolution Microscopy

Photoinduced off-switching of organic fluorophores is routinely used in super-resolution microscopy to separate and localize single fluorescent molecules, but the method typically relies on the use of complex imaging buffers. The most common buffers use primary thiols to reversibly reduce excited fluorophores to a non-fluorescent dark state, but these thiols have a limited shelf life and additionally require high illumination intensities in order to efficiently switch the emission of fluorophores. Recently a high-index, thiol-containing imaging buffer emerged which used sodium sulfite as an oxygen scavenger, but the switching properties of sulfite was not reported on. Here, we show that sodium sulfite in common buffer solutions reacts with fluorescent dyes, such as Alexa Fluor 647 and Alexa Fluor 488 under low to medium intensity illumination to form a semi-stable dark state. The duration of this dark state can be tuned by adding glycerol to the buffer. This simplifies the realization of different super-resolution microscopy modalities such as direct Stochastic Reconstruction Microscopy (dSTORM) and Super-resolution Optical Fluctuation Microscopy (SOFI). We characterize sulfite as a switching agent and compare it to the two most common switching agents by imaging cytoskeleton structures such as microtubules and the actin cytoskeleton in human osteosarcoma cells.

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 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 Towards a Quantitative Single Particle Characterization by Super Resolution Microscopy: From Virus Structures to Antivirals Design

2021 ◽  
Vol 9 ◽  
Author(s):  
Maria Arista-Romero ◽  
Silvia Pujals ◽  
Lorenzo Albertazzi
Keyword(s):  
Viral Infection ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Particle Characterization ◽  
Emerging Viruses ◽  
Potential Threat ◽  
The Right ◽  
Super Resolution Microscopy ◽  
Selection Of

In the last year the COVID19 pandemic clearly illustrated the potential threat that viruses pose to our society. The characterization of viral structures and the identification of key proteins involved in each step of the cycle of infection are crucial to develop treatments. However, the small size of viruses, invisible under conventional fluorescence microscopy, make it difficult to study the organization of protein clusters within the viral particle. The applications of super-resolution microscopy have skyrocketed in the last years, converting this group into one of the leading techniques to characterize viruses and study the viral infection in cells, breaking the diffraction limit by achieving resolutions up to 10 nm using conventional probes such as fluorescent dyes and proteins. There are several super-resolution methods available and the selection of the right one it is crucial to study in detail all the steps involved in the viral infection, quantifying and creating models of infection for relevant viruses such as HIV-1, Influenza, herpesvirus or SARS-CoV-1. Here we review the use of super-resolution microscopy (SRM) to study all steps involved in the viral infection and antiviral design. In light of the threat of new viruses, these studies could inspire future assays to unveil the viral mechanism of emerging viruses and further develop successful antivirals against them.

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 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 Super-resolution microscopy reveals that disruption of ciliary transition zone architecture is a cause of Joubert syndrome

2017 ◽  
Author(s):  
Xiaoyu Shi ◽  
Galo Garcia ◽  
Julie C. Van De Weghe ◽  
Ryan McGorty ◽  
Gregory J. Pazour ◽  
...  
Keyword(s):  
Transition Zone ◽  
Human Fibroblasts ◽  
Super Resolution ◽  
Protein Composition ◽  
Joubert Syndrome ◽  
Complex Component ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Developmental Signaling ◽  
Optical Reconstruction ◽  
Super Resolution Microscopy

ABSTRACTDiverse human ciliopathies, including nephronophthisis (NPHP), Meckel syndrome (MKS) and Joubert syndrome (JBTS), can be caused by mutations affecting components of the transition zone, a ciliary domain near its base. The transition zone controls the protein composition of the ciliary membrane, but how it does so is unclear. To better understand the transition zone and its connection to ciliopathies, we defined the arrangement of key proteins in the transition zone using two-color stochastic optical reconstruction microscopy (STORM). This mapping revealed that NPHP and MKS complex components form nested rings comprised of nine-fold doublets. The NPHP complex component RPGRIP1L forms a smaller diameter transition zone ring within the MKS complex rings. JBTS-associated mutations in RPGRIP1L disrupt the architecture of the MKS and NPHP rings, revealing that vertebrate RPGRIP1L has a key role in organizing transition zone architecture. JBTS-associated mutations in TCTN2, encoding an MKS complex component, also displace proteins of the MKS and NPHP complexes from the transition zone, revealing that RPGRIP1L and TCTN2 have interdependent roles in organizing transition zone architecture. To understand how altered transition zone architecture affects developmental signaling, we examined the localization of the Hedgehog pathway component SMO in human fibroblasts derived from JBTS-affected individuals. We found that diverse ciliary proteins, including SMO, accumulate at the transition zone in wild type cells, suggesting that the transition zone is a way station for proteins entering and exiting the cilium. JBTS-associated mutations in RPGRIP1L disrupt SMO accumulation at the transition zone and the ciliary localization of SMO. We propose that the disruption of transition zone architecture in JBTS leads to a failure of SMO to accumulate at the transition zone, disrupting developmental signaling in JBTS.

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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