scholarly journals Unraveling the Nanoscopic Organization and Function of Central Mammalian Presynapses With Super-Resolution Microscopy

2021 ◽  
Vol 14 ◽  
Author(s):  
Lia G. Carvalhais ◽  
Vera C. Martinho ◽  
Elisabete Ferreiro ◽  
Paulo S. Pinheiro
Keyword(s):  
Imaging Techniques ◽  
Rapid Evolution ◽  
Super Resolution ◽  
Synaptic Function ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Axon Terminals ◽  
Optical Reconstruction ◽  
And Function

The complex, nanoscopic scale of neuronal function, taking place at dendritic spines, axon terminals, and other minuscule structures, cannot be adequately resolved using standard, diffraction-limited imaging techniques. The last couple of decades saw a rapid evolution of imaging methods that overcome the diffraction limit imposed by Abbe’s principle. These techniques, including structured illumination microscopy (SIM), stimulated emission depletion (STED), photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM), among others, have revolutionized our understanding of synapse biology. By exploiting the stochastic nature of fluorophore light/dark states or non-linearities in the interaction of fluorophores with light, by using modified illumination strategies that limit the excitation area, these methods can achieve spatial resolutions down to just a few tens of nm or less. Here, we review how these advanced imaging techniques have contributed to unprecedented insight into the nanoscopic organization and function of mammalian neuronal presynapses, revealing new organizational principles or lending support to existing views, while raising many important new questions. We further discuss recent technical refinements and newly developed tools that will continue to expand our ability to delve deeper into how synaptic function is orchestrated at the nanoscopic level.

scholarly journals Super-Resolution Imaging of Tight and Adherens Junctions: Challenges and Open Questions

2020 ◽  
Vol 21 (3) ◽  
pp. 744 ◽  
Author(s):  
Hannes Gonschior ◽  
Volker Haucke ◽  
Martin Lehmann
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Rapid Development ◽  
Ion Homeostasis ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Molecular Architecture ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging

The tight junction (TJ) and the adherens junction (AJ) bridge the paracellular cleft of epithelial and endothelial cells. In addition to their role as protective barriers against bacteria and their toxins they maintain ion homeostasis, cell polarity, and mechano-sensing. Their functional loss leads to pathological changes such as tissue inflammation, ion imbalance, and cancer. To better understand the consequences of such malfunctions, the junctional nanoarchitecture is of great importance since it remains so far largely unresolved, mainly because of major difficulties in dynamically imaging these structures at sufficient resolution and with molecular precision. The rapid development of super-resolution imaging techniques ranging from structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (SMLM) has now enabled molecular imaging of biological specimens from cells to tissues with nanometer resolution. Here we summarize these techniques and their application to the dissection of the nanoscale molecular architecture of TJs and AJs. We propose that super-resolution imaging together with advances in genome engineering and functional analyses approaches will create a leap in our understanding of the composition, assembly, and function of TJs and AJs at the nanoscale and, thereby, enable a mechanistic understanding of their dysfunction in disease.

scholarly journals Super-Resolved Fluorescence Imaging of Peripheral Nerve  

2021 ◽  
Author(s):  
Iván Coto Hernández ◽  
Suresh Mohan ◽  
Nate Jowett
Keyword(s):  
Peripheral Nerve ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Biomedical Science ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Unmyelinated Axons ◽  
Histopathologic Evaluation ◽  
Resolution Imaging

Abstract Traditional histopathologic evaluation of peripheral nerve employs brightfield microscopy with diffraction limited resolution of ~ 250 nm. Though electron microscopy yields nanoscale resolution of the nervous system, it is resource-intensive and incompatible with life. Super-resolution microscopy (SRM) comprises a set of imaging techniques permitting unprecedented resolution of fluorescent objects using visible light. The advent of SRM has transformed biomedical science in establishing non-toxic means for investigation of nanoscale cellular structures. Herein, sciatic nerve sections from GFP-variant expressing mice, and regenerating human nerve from cross-facial autografts labelled with a myelin-specific fluorescent dye were imaged by super-resolution radial fluctuation microscopy, stimulated emission depletion microscopy, and structured illumination microscopy. Super-resolution imaging of axial cryosections of murine sciatic nerves demonstrated robust visualization of myelinated and unmyelinated axons. Super-resolution imaging of axial cryosections of human cross-facial nerve grafts demonstrated enhanced resolution of small-calibre thinly-myelinated regenerating motor axons. The utility of SRM in imaging of mammalian cranial and peripheral nerves is demonstrated. The increase in contrast and structural clarity achievable with super-resolution techniques enables visualization of unmyelinated axons, regenerating axons, cytoskeleton ultrastructure, and neuronal appendages using light microscopes.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

Super-Resolution Imaging in Fluid Mechanics Using New Illumination Approaches

2020 ◽  
Vol 52 (1) ◽  
pp. 369-393
Author(s):  
Minami Yoda
Keyword(s):  
Fluid Mechanics ◽  
Total Internal Reflection ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Internal Reflection ◽  
Interfacial Flows ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Entire Volume ◽  
Resolution Imaging

Quantifying submillimeter flows using optical diagnostic techniques is often limited by a lack of spatial resolution and optical access. This review discusses two super-resolution imaging techniques, structured illumination microscopy and total internal reflection fluorescence or microscopy, which can visualize bulk and interfacial flows, respectively, at spatial resolutions below the classic diffraction limits. First, we discuss the theory and applications of structured illumination for optical sectioning, i.e., imaging a thin slice of a flow illuminated over its entire volume. Structured illumination can be used to visualize the interior of multiphase flows such as sprays by greatly reducing secondary scattering. Second, the theory underlying evanescent waves is introduced, followed by a review of how total internal reflection microscopy has been used to visualize interfacial flows over the last 15 years. Both techniques, which are starting to be used in fluid mechanics, could significantly improve quantitative imaging of microscale and macroscale flows.

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 A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies

2020 ◽  
Vol 12 (535) ◽  
pp. eaay0071 ◽  
Author(s):  
Zhen Liu ◽  
Quynh P. H. Nguyen ◽  
Qingxu Guan ◽  
Alexandra Albulescu ◽  
Lauren Erdman ◽  
...  
Keyword(s):  
Lung Function ◽  
A Priori ◽  
Three Dimensional ◽  
Super Resolution ◽  
Structural Defects ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Motile Cilia ◽  
Optical Reconstruction

Airway clearance of pathogens and particulates relies on motile cilia. Impaired cilia motility can lead to reduction in lung function, lung transplant, or death in some cases. More than 50 proteins regulating cilia motility are linked to primary ciliary dyskinesia (PCD), a heterogeneous, mainly recessive genetic lung disease. Accurate PCD molecular diagnosis is essential for identifying therapeutic targets and for initiating therapies that can stabilize lung function, thereby reducing socioeconomic impact of the disease. To date, PCD diagnosis has mainly relied on nonquantitative methods that have limited sensitivity or require a priori knowledge of the genes involved. Here, we developed a quantitative super-resolution microscopy workflow: (i) to increase sensitivity and throughput, (ii) to detect structural defects in PCD patients’ cells, and (iii) to quantify motility defects caused by yet to be found PCD genes. Toward these goals, we built a localization map of PCD proteins by three-dimensional structured illumination microscopy and implemented quantitative image analysis and machine learning to detect protein mislocalization, we analyzed axonemal structure by stochastic optical reconstruction microscopy, and we developed a high-throughput method for detecting motile cilia uncoordination by rotational polarity. Together, our data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.

scholarly journals Laser-free super-resolution microscopy

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Kirti Prakash
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Correlative Imaging ◽  
Deep Blue ◽  
Meiotic Chromosomes ◽  
Set Up ◽  
Super Resolution Microscopy

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope set-up and a mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localization microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as ‘blinking’), detected and localized. The use of a short burst of deep blue excitation (350–380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that stimulated emission depletion and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

scholarly journals Effect of light polariztion on pattern illumination super-resolution imaging

2016 ◽  
Vol 09 (03) ◽  
pp. 1641001 ◽  
Author(s):  
Caimin Qiu ◽  
Jianling Chen ◽  
Zexian Hou ◽  
Chaoxian Xu ◽  
Shusen Xie ◽  
...  
Keyword(s):  
Polarized Light ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Excitation Light ◽  
Imaging Technology ◽  
Circularly Polarized Light ◽  
Resolution Imaging ◽  
The Right

Far-field fluorescence microscopy has made great progress in the spatial resolution, limited by light diffraction, since the super-resolution imaging technology appeared. And stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) can be grouped into one class of the super-resolution imaging technology, which use pattern illumination strategy to circumvent the diffraction limit. We simulated the images of the beads of SIM imaging, the intensity distribution of STED excitation light and depletion light in order to observe effects of the polarized light on imaging quality. Compared to fixed linear polarization, circularly polarized light is more suitable for SIM on reconstructed image. And right-handed circular polarization (CP) light is more appropriate for both the excitation and depletion light in STED system. Therefore the right-handed CP light would be the best candidate when the SIM and STED are combined into one microscope. Good understanding of the polarization will provide a reference for the patterned illumination experiment to achieve better resolution and better image quality.

scholarly journals CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication

2019 ◽  
Vol 159 (1) ◽  
pp. 48-53 ◽  
Author(s):  
Alžběta Němečková ◽  
Christina Wäsch ◽  
Veit Schubert ◽  
Takayoshi Ishii ◽  
Eva Hřibová ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromatin Structure ◽  
Super Resolution ◽  
Dna Repeats ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Replication Sites ◽  
And Function ◽  
Super Resolution Microscopy

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.

scholarly journals Structured illumination microscopy and its new developments

2016 ◽  
Vol 09 (03) ◽  
pp. 1630010 ◽  
Author(s):  
Jianling Chen ◽  
Caimin Qiu ◽  
Minghai You ◽  
Xiaogang Chen ◽  
Hongqin Yang ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Spatial Resolution ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Living Cells ◽  
The Other ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Recent Developments ◽  
Super Resolution Microscopy

Optical microscopy allows us to observe the biological structures and processes within living cells. However, the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light diffraction. Structured illumination microscopy (SIM), a type of new emerging super-resolution microscopy, doubles the spatial resolution by illuminating the specimen with a patterned light, and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy. In addition, SIM is easier to combine with the other imaging techniques to improve their imaging resolution, leading to the developments of diverse types of SIM. SIM has great potential to meet the various requirements of living cells imaging. Here, we review the recent developments of SIM and its combination with other imaging techniques.

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