scholarly journals 3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

2019 ◽  
Author(s):  
Ruisheng Lin ◽  
Alexander H. Clowsley ◽  
Tobias Lutz ◽  
David Baddeley ◽  
Christian Soeller
Keyword(s):  
Quantitative Imaging ◽  
Super Resolution ◽  
Cost Effective ◽  
Dna Origami ◽  
Robust Test ◽  
Imaging Quality ◽  
Multiple Test ◽  
Resolution Imaging ◽  
2D Imaging ◽  
Super Resolution Microscopy

AbstractAssessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time-and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.

scholarly journals Label-free super-resolution imaging below 90-nm using photon-reassignment

2021 ◽  
Vol 1 ◽  
pp. 3
Author(s):  
Alberto Aguilar ◽  
Adeline Boyreau ◽  
Pierre Bon
Keyword(s):  
Biological Samples ◽  
Optical Resolution ◽  
Super Resolution ◽  
Cost Effective ◽  
Label Free ◽  
Cost Effective Approach ◽  
Imaging Mode ◽  
Resolution Imaging ◽  
Fluorescent Tags ◽  
Super Resolution Microscopy

Background: Achieving resolutions below 100 nm is key for many fields, including biology and nanomaterial characterization. Although nearfield and electron microscopy are the gold standards for studying the nanoscale, optical microscopy has seen its resolution drastically improve in the last decades. So-called super-resolution microscopy is generally based on fluorescence photophysics and requires modification of the sample at least by adding fluorescent tags, an inevitably invasive step. Therefore, it remains very challenging and rewarding to achieve optical resolutions beyond the diffraction limit in label-free samples. Methods: Here, we present a breakthrough to unlock label-free 3D super-resolution imaging of any object including living biological samples. It is based on optical photon-reassignment in confocal reflectance imaging mode. Results: We demonstrate that we surpass the resolution of all fluorescence-based confocal systems by a factor ~1.5. We have obtained images with a 3D (x,y,z) optical resolution of (86x86x248) nm3 using a visible wavelength (445 nm) and a regular microscope objective (NA=1.3). The results are presented on nanoparticles as well as on (living) biological samples. Conclusions: This cost-effective approach double the resolution of reflectance confocal microscope with minimal modifications. It is therefore compatible with any microscope and sample, works in real-time, and does not require any signal processing.

scholarly journals AN ECONOMIC, SQUARE-SHAPED FLAT-FIELD ILLUMINATION MODULE FOR TIRF-BASED SUPER-RESOLUTION MICROSCOPY

2021 ◽  
Author(s):  
Jeff Y L Lam ◽  
Yunzhao Wu ◽  
Eleni Dimou ◽  
Ziwei Zhang ◽  
Matthew R Cheetham ◽  
...  
Keyword(s):  
High Throughput ◽  
Total Internal Reflection ◽  
Quantitative Imaging ◽  
Super Resolution ◽  
Cost Effective ◽  
Cellular Systems ◽  
Cost Effective Method ◽  
Flat Field ◽  
Super Resolution Microscopy ◽  
Highly Uniform

Super-resolution (SR) microscopy allows complex biological assemblies to be observed with remarkable resolution. However, the presence of uneven Gaussian-shaped illumination hinders its use in quantitative imaging or high-throughput assays. Methods developed to circumvent this problem are often expensive, hard-to-implement, or not applicable to total internal reflection fluorescence (TIRF) imaging. We herein demonstrate a cost-effective method to overcome these challenges using a small square-core multimodal optical fibre as the coupler. We characterise our method with synthetic, recombinant and cellular systems imaged under TIRF and highly inclined and laminated optical sheet (HILO) illuminations to demonstrate its ability to produce highly uniform images under all conditions.

scholarly journals Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells

Nanoscale ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 10023-10033 ◽  
Author(s):  
Jan Bergstrand ◽  
Lei Xu ◽  
Xinyan Miao ◽  
Nailin Li ◽  
Ozan Öktem ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Dictionary Learning ◽  
Super Resolution ◽  
Distribution Patterns ◽  
Specific Protein ◽  
Protein Distribution ◽  
Activated Platelets ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Super-resolution imaging of P-selectin in platelets together with dictionary learning allow specifically activated platelets to be identified in an automatic objective manner.

scholarly journals 3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

Methods ◽  
2020 ◽  
Vol 174 ◽  
pp. 56-71 ◽  
Author(s):  
Ruisheng Lin ◽  
Alexander H. Clowsley ◽  
Tobias Lutz ◽  
David Baddeley ◽  
Christian Soeller
Keyword(s):  
Quantitative Analysis ◽  
Super Resolution ◽  
Dna Origami ◽  
Super Resolution Microscopy

scholarly journals Super-Resolution Microscopy of Chromatin

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 493 ◽  
Author(s):  
Birk
Keyword(s):  
Gene Regulation ◽  
Data Analysis ◽  
Super Resolution ◽  
Fluorescence Labeling ◽  
Cellular Processes ◽  
Direct Assessment ◽  
Chromatin Interactions ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Insight Into

Since the advent of super-resolution microscopy, countless approaches and studies have been published contributing significantly to our understanding of cellular processes. With the aid of chromatin-specific fluorescence labeling techniques, we are gaining increasing insight into gene regulation and chromatin organization. Combined with super-resolution imaging and data analysis, these labeling techniques enable direct assessment not only of chromatin interactions but also of the function of specific chromatin conformational states.

scholarly journals Bioorthogonal double-fluorogenic siliconrhodamine probes for intracellular super-resolution microscopy

2017 ◽  
Vol 53 (50) ◽  
pp. 6696-6699 ◽  
Author(s):  
E. Kozma ◽  
G. Estrada Girona ◽  
G. Paci ◽  
E. A. Lemke ◽  
P. Kele
Keyword(s):  
Super Resolution ◽  
Site Specific ◽  
Intracellular Proteins ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

A series of double-fluorogenic siliconrhodamine-tetrazines were synthesized. One of these tetrazines is a membrane-permeant label allowing site-specific bioorthogonal tagging of intracellular proteins and super-resolution imaging.

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.

Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami

Nano Letters ◽  
2010 ◽  
Vol 10 (11) ◽  
pp. 4756-4761 ◽  
Author(s):  
Ralf Jungmann ◽  
Christian Steinhauer ◽  
Max Scheible ◽  
Anton Kuzyk ◽  
Philip Tinnefeld ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Super Resolution ◽  
Dna Origami ◽  
Super Resolution Microscopy

scholarly journals Quantitative comparison of camera technologies for cost-effective Super-resolution Optical Fluctuation Imaging (SOFI)

2018 ◽  
Author(s):  
Robin Van den Eynde ◽  
Alice Sandmeyer ◽  
Wim Vandenberg ◽  
Sam Duwé ◽  
Wolfgang Hübner ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Imaging System ◽  
Fluorescent Proteins ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Cost Effective ◽  
Wide Field ◽  
Cmos Camera ◽  
Resolution Imaging ◽  
Cost Efficient

AbstractSuper-Resolution (SR) fluorescence microscopy is typically carried out on high-end research microscopes. Super-resolution Optical Fluctuation Imaging (SOFI) is a fast SR technique capable of live-cell imaging, that is compatible with many wide-field microscope systems. However, especially when employing fluorescent proteins, a key part of the imaging system is a very sensitive and well calibrated camera sensor. The substantial costs of such systems preclude many research groups from employing super-resolution imaging techniques.Here, we examine to what extent SOFI can be performed using a range of imaging hardware comprising different technologies and costs. In particular, we quantitatively compare the performance of an industry-grade CMOS camera to both state-of-the-art emCCD and sCMOS detectors, with SOFI-specific metrics. We show that SOFI data can be obtained using a cost-efficient industry-grade sensor, both on commercial and home-built microscope systems, though our analysis also readily exposes the merits of the per-pixel corrections performed in scientific cameras.

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.

Sign in / Sign up

Export Citation Format

Share Document