scholarly journals UCsim2: 2D Structured Illumination Microscopy using UC2

2021 ◽  
Author(s):  
Haoran Wang ◽  
Réne Lachmann ◽  
Barbora Marsikova ◽  
Rainer Heinzmann ◽  
Benedict Diederich
Keyword(s):  
Open Source ◽  
Super Resolution ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
Manufacturing Tolerance ◽  
Resolution Imaging ◽  
Open Source Hardware ◽  
Microscopy Techniques ◽  
Diffraction Barrier

State-of-the-art microscopy techniques enable the imaging of sub-diffraction barrier biological structures at the price of high-costs or lacking transparency. We try to reduce some of these barriers by presenting a super-resolution upgrade to our recently presented open-source optical toolbox UC2. Our new injection moulded parts allow larger builds with higher precision. The 4x lower manufacturing tolerance compared to 3D printing makes assemblies more reproducible. By adding consumer-grade available open-source hardware such as digital mirror devices (DMD) and laser projectors we demonstrate a compact 3D multimodal setup that combines image scanning microscopy (ISM) and structured illumination microscopy (SIM). We demonstrate a gain in resolution and optical sectioning using the two different modes compared to the widefield limit by imaging Alexa Fluor 647- and SiR-stained HeLa cells. We compare different objective lenses and by sharing the designs and manuals of our setup, we make super-resolution imaging available to everyone.

scholarly journals Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction

2018 ◽  
Author(s):  
Jakub Pospíšil ◽  
Tomáš Lukeš ◽  
Justin Bendesky ◽  
Karel Fliegel ◽  
Kathrin Spendier ◽  
...  
Keyword(s):  
High Resolution ◽  
Open Source ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Live Cells ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
Raw Data ◽  
Bayesian Image Reconstruction

AbstractBackgroundStructured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution).FindingsFive complete and freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods, and with newer Bayesian restoration approaches which we are developing.ConclusionVarious methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments is not typically published. Publicly available, high quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data was processed with SIMToolbox, an open source and freely available software solution for SIM.

scholarly journals Super-resolution structured illumination microscopy: past, present and future

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Kirti Prakash ◽  
Benedict Diederich ◽  
Stefanie Reichelt ◽  
Rainer Heintzmann ◽  
Lothar Schermelleh
Keyword(s):  
Three Dimensional ◽  
Super Resolution ◽  
Computational Imaging ◽  
Biological Applications ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Recent Developments ◽  
Resolution Imaging ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

Structured illumination microscopy (SIM) has emerged as an essential technique for three-dimensional (3D) and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualization and image processing methods.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

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 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 Artifact-free whole-slide imaging with structured illumination microscopy and Bayesian image reconstruction

GigaScience ◽  
2020 ◽  
Vol 9 (4) ◽  
Author(s):  
Karl A Johnson ◽  
Guy M Hagen
Keyword(s):  
Super Resolution ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
High Quality ◽  
Related Data ◽  
Reconstruction Methods ◽  
Set Up ◽  
Bayesian Image Reconstruction ◽  
Hematoxylin Eosin

Abstract Background Structured illumination microscopy (SIM) is a method that can be used to image biological samples and can achieve both optical sectioning and super-resolution effects. Optimization of the imaging set-up and data-processing methods results in high-quality images without artifacts due to mosaicking or due to the use of SIM methods. Reconstruction methods based on Bayesian estimation can be used to produce images with a resolution beyond that dictated by the optical system. Findings Five complete datasets are presented including large panoramic SIM images of human tissues in pathophysiological conditions. Cancers of the prostate, skin, ovary, and breast, as well as tuberculosis of the lung, were imaged using SIM. The samples are available commercially and are standard histological preparations stained with hematoxylin-eosin. Conclusion The use of fluorescence microscopy is increasing in histopathology. There is a need for methods that reduce artifacts caused by the use of image-stitching methods or optical sectioning methods such as SIM. Stitched SIM images produce results that may be useful for intraoperative histology. Releasing high-quality, full-slide images and related data will aid researchers in furthering the field of fluorescent histopathology.

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