scholarly journals High Numerical Aperture Epi-illumination Selective Plane Illumination Microscopy

2018 ◽  
Author(s):  
Bin Yang ◽  
Yina Wang ◽  
Siyu Feng ◽  
Veronica Pessino ◽  
Nico Stuurman ◽  
...  
Keyword(s):  
Single Molecule ◽  
Numerical Aperture ◽  
Super Resolution ◽  
Live Cells ◽  
Volumetric Imaging ◽  
High Numerical Aperture ◽  
Selective Plane Illumination Microscopy ◽  
Resolution Imaging ◽  
Living Organisms ◽  
Super Resolution Microscopy

Selective-plane illumination microscopy (SPIM) provides unparalleled advantages for long-term volumetric imaging of living organisms. In order to achieve high-resolution imaging in common biological sample holders, we designed a high numerical aperture (NA) epi-illumination SPIM (eSPIM) system, which utilizes a single objective and has an identical sample interface as an inverted fluorescence microscope with no additional reflection elements. This system has an effective detection NA of > 1.06. We demonstrated multicolor and fast volumetric imaging of live cells and single-molecule super-resolution microscopy using our system.

scholarly journals Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

2020 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Sebastian Reinhard ◽  
Davide Gambarotto ◽  
Toby D. M. Bell ◽  
Virginie Hamel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Reference Structure ◽  
Positional Error ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

scholarly journals 3D Interferometric Lattice Light-Sheet Imaging

2020 ◽  
Author(s):  
Bin Cao ◽  
Guanshi Wang ◽  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Detection Sensitivity ◽  
Live Cells ◽  
Background Suppression ◽  
Axial Resolution ◽  
Volumetric Imaging ◽  
Spatio Temporal ◽  
And Function

Understanding cellular structure and function requires live-cell imaging with high spatio-temporal resolution and high detection sensitivity. Direct visualization of molecular processes using single-molecule/super-resolution techniques has thus been transformative. However, extracting the highest-resolution 4D information possible from weak and dynamic fluorescence signals in live cells remains challenging. For example, some of the highest spatial resolution methods, e.g. interferometric (4Pi) approaches1-6 can be slow, require high peak excitation intensities that accelerate photobleaching or suffer from increased out-of-focus background. Selective-plane illumination (SPIM)7-12 reduces background, but most implementations typically feature modest spatial, especially axial, resolution. Here we develop 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that overcomes many of these limitations. 3D-iLLS provides, by virtue of SPIM, low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. We demonstrate 3D-iLLS with axial resolution and single-particle localization precision down to <100nm (FWHM) and <10nm (1σ) respectively. 3D-iLLS paves the way for a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and SNR live imaging.

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach

2018 ◽  
Vol 20 (12) ◽  
pp. 8088-8098 ◽  
Author(s):  
Rajeev Yadav ◽  
H. Peter Lu
Keyword(s):  
Ion Channel ◽  
Single Molecule ◽  
Super Resolution ◽  
Live Cells ◽  
Imaging Approach ◽  
Resolution Imaging ◽  
Function Relationship ◽  
Bleaching Step ◽  
Relationship Of ◽  
Resolution Single

Correlating single-molecule fluorescence photo-bleaching step analysis and single-molecule super-resolution imaging, our findings for the clustering effect of the NMDA receptor ion channel on the live cell membranes provide a new and significant understanding of the structure–function relationship of NMDA receptors.

scholarly journals Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrew Tze Fui Liew ◽  
Yong Hwee Foo ◽  
Yunfeng Gao ◽  
Parisa Zangoui ◽  
Moirangthem Kiran Singh ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Single Cells ◽  
Super Resolution ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Bacterial Cells ◽  
Acid Ph ◽  
Resolution Imaging ◽  
Ph Dependent

After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella.

scholarly journals Nanoscopy on the Chea(i)p

2020 ◽  
Author(s):  
Benedict Diederich ◽  
Øystein Helle ◽  
Patrick Then ◽  
Pablo Carravilla ◽  
Kay Oliver Schink ◽  
...  
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Low Cost ◽  
Super Resolution ◽  
Image Sensor ◽  
Optical Diffraction ◽  
Sensor Technology ◽  
Safety Level ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

AbstractSuper-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.

Super-resolution microscopy of live cells using single molecule localization

2013 ◽  
Vol 58 (36) ◽  
pp. 4519-4527 ◽  
Author(s):  
YongDeng Zhang ◽  
Hao Chang ◽  
LuSheng Gu ◽  
YanHua Zhao ◽  
Tao Xu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Live Cells ◽  
Super Resolution Microscopy

scholarly journals Drosophila Models Rediscovered with Super-Resolution Microscopy

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1924
Author(s):  
Szilárd Szikora ◽  
Péter Görög ◽  
Csaba Kozma ◽  
József Mihály
Keyword(s):  
Classical Model ◽  
Super Resolution ◽  
Model Systems ◽  
Model Organisms ◽  
Biological Phenomena ◽  
Drosophila Model ◽  
Resolution Imaging ◽  
Living Organisms ◽  
Super Resolution Microscopy ◽  
Immense Potential

With the advent of super-resolution microscopy, we gained a powerful toolbox to bridge the gap between the cellular- and molecular-level analysis of living organisms. Although nanoscopy is broadly applicable, classical model organisms, such as fruit flies, worms and mice, remained the leading subjects because combining the strength of sophisticated genetics, biochemistry and electrophysiology with the unparalleled resolution provided by super-resolution imaging appears as one of the most efficient approaches to understanding the basic cell biological questions and the molecular complexity of life. Here, we summarize the major nanoscopic techniques and illustrate how these approaches were used in Drosophila model systems to revisit a series of well-known cell biological phenomena. These investigations clearly demonstrate that instead of simply achieving an improvement in image quality, nanoscopy goes far beyond with its immense potential to discover novel structural and mechanistic aspects. With the examples of synaptic active zones, centrosomes and sarcomeres, we will explain the instrumental role of super-resolution imaging pioneered in Drosophila in understanding fundamental subcellular constituents.

scholarly journals High-NA open-top selective-plane illumination microscopy for biological imaging

2017 ◽  
Author(s):  
Ryan McGorty ◽  
Dan Xie ◽  
Bo Huang
Keyword(s):  
Adaptive Optics ◽  
Biological Sample ◽  
Spatial Configuration ◽  
Numerical Aperture ◽  
Biological Imaging ◽  
Imaging Systems ◽  
Optical Components ◽  
Volumetric Imaging ◽  
Selective Plane Illumination Microscopy ◽  
Living Organisms

Abstract:Selective-plane illumination microscopy (SPIM) provides unparalleled advantages for volumetric imaging of living organisms over extended times. However, the spatial configuration of a SPIM system often limits its compatibility with many widely used biological sample holders such as multi-well chambers and plates. To solve this problem, we developed a high numerical aperture (NA) open-top configuration that places both the excitation and detection objectives on the opposite of the sample coverglass. We carried out a theoretical calculation to analyze the structure of the system-induced aberrations. We then experimentally compensated the system aberrations using adaptive optics combined with static optical components, demonstrating near-diffraction-limited performance in imaging fluorescently labeled cells.© 2017 Optical Society of AmericaOCIS codes: (080.080) Geometric Optics; (110.0110) Imaging systems; (110.0180) Microscopy.

scholarly journals Fast DNA-PAINT imaging using a deep neural network

2021 ◽  
Author(s):  
Kaarjel K Narayanasamy ◽  
Johanna V Rahm ◽  
Siddharth Tourani ◽  
Mike Heilemann
Keyword(s):  
Neural Network ◽  
Single Molecule ◽  
Image Acquisition ◽  
Super Resolution ◽  
Large Samples ◽  
The Neural Network ◽  
Target Imaging ◽  
Nanoscale Topography ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique with relatively easy-to-implement multi-target imaging. However, image acquisition is slow as sufficient statistical data has to be generated from spatio-temporally isolated single emitters. Here, we trained the neural network (NN) DeepSTORM to predict fluorophore positions from high emitter density DNA-PAINT data. This achieves image acquisition in one minute. We demonstrate multi-color super-resolution imaging of structure-conserved semi-thin neuronal tissue and imaging of large samples. This improvement can be integrated into any single-molecule microscope and enables fast single-molecule super-resolution microscopy.

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