scholarly journals Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ralph Götz ◽  
Tobias C. Kunz ◽  
Julian Fink ◽  
Franziska Solger ◽  
Jan Schlegel ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Bacterial Infections ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Nanoscale Imaging ◽  
Intracellular Organelles ◽  
Resolution Imaging ◽  
Simkania Negevensis ◽  
20 Nm

AbstractExpansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

scholarly journals Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

2020 ◽  
Author(s):  
Ralph Götz ◽  
Tobias C. Kunz ◽  
Julian Fink ◽  
Franziska Solger ◽  
Jan Schlegel ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Bacterial Infections ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Nanoscale Imaging ◽  
Intracellular Organelles ◽  
Resolution Imaging ◽  
Simkania Negevensis ◽  
20 Nm

AbstractExpansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4x to 10x expansion. Confocal and structured illumination microscopy (SIM) enabled imaging of sphingolipids and their interactions with proteins in the membrane of intracellular organelles with a spatial resolution of 10-20 nm. Because sphingolipids accumulated efficiently in pathogens we used sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allowed us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

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.

scholarly journals Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Karl Zhanghao ◽  
Xingye Chen ◽  
Wenhui Liu ◽  
Meiqi Li ◽  
Yiqiong Liu ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Imaging Modality ◽  
Super Resolution ◽  
Vital Role ◽  
Molecular Structures ◽  
Polarization Microscopy ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging

Abstract Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

scholarly journals Super-Resolution Imaging by Dual Iterative Structured Illumination Microscopy

2021 ◽  
Author(s):  
Anna Loeschberger ◽  
Yauheni Novikau ◽  
Ralf Netz ◽  
Marie-Christin Spindler ◽  
Ricardo Benavente ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Brain Slices ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution ◽  
Coated Pits ◽  
Resolution Imaging

Three-dimensional (3D) multicolor super-resolution imaging in the 50-100 nm range in fixed and living cells remains challenging. We extend the resolution of structured illumination microscopy (SIM) by an improved nonlinear iterative reconstruction algorithm that enables 3D multicolor imaging with improved spatiotemporal resolution at low illumination intensities. We demonstrate the performance of dual iterative SIM (diSIM) imaging cellular structures in fixed cells including synaptonemal complexes, clathrin coated pits and the actin cytoskeleton with lateral resolutions of 60-100 nm with standard fluorophores. Furthermore, we visualize dendritic spines in 70 micrometer thick brain slices with an axial resolution < 200 nm. Finally, we image dynamics of the endoplasmatic reticulum and microtubules in living cells with up to 255 frames/s.

scholarly journals Tilt illumination for structured illumination imaging

2021 ◽  
Author(s):  
Xin Jin ◽  
Xuemei Ding ◽  
Jiubin Tan ◽  
Cheng Shen ◽  
Xuyang Zhou ◽  
...  
Keyword(s):  
Transfer Function ◽  
Super Resolution ◽  
Lateral Resolution ◽  
Optical Transfer Function ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Sample Spectrum ◽  
Resolution Imaging ◽  
Optical Transfer ◽  
Illumination Mode

Abstract To achieve super-resolution imaging, the information in higher frequency of the observed sample is collected by illuminating with a structure beam for a limited optical transfer function (OTF). In this paper, tilt illumination mode is introduced to structured illumination microscopy (SIM) for enhancing lateral resolution. More sample spectrum more than traditional SIM, can be obtained by detector. Thus, SIM with tilt illumination can be improved at the aspect of lateral imaging resolution.

scholarly journals Applications of Super Resolution Expansion Microscopy in Yeast

2021 ◽  
Vol 9 ◽  
Author(s):  
Liwen Chen ◽  
Longfang Yao ◽  
Li Zhang ◽  
Yiyan Fei ◽  
Lan Mi ◽  
...  
Keyword(s):  
Super Resolution ◽  
Imaging Study ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Preparation Technique ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging ◽  
Conventional Microscopy ◽  
Related Proteins

Super-resolution microscopy includes multiple techniques in optical microscopy that enable sub-diffraction resolution fluorescence imaging of cellular structures. Expansion microscopy (EXM) is a method of physical expansion to obtain super-resolution images of a biological sample on conventional microscopy. We present images of yeast organelles, applying the combination of super-resolution and ExM techniques. When preparing pre-expanded samples, conventional methods lead to breakage of dividing yeast cells and difficulties in studying division-related proteins. Here, we describe an improved sample preparation technique that avoids such damage. ExM in combination with Airyscan and structured illumination microscopy (SIM) collected sub-cellular structural images of nuclear pore complex, septin, and a-tubulin in yeast. Our method of expansion in yeast is well-suited for super-resolution imaging study of yeast.

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