scholarly journals Sparse deconvolution of high-density super-resolution images

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Siewert Hugelier ◽  
Johan J. de Rooi ◽  
Romain Bernex ◽  
Sam Duwé ◽  
Olivier Devos ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Super Resolution ◽  
Penalized Regression ◽  
High Density ◽  
Wide Field ◽  
Cellular Processes ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Simulated Images ◽  
Norm Penalty

Abstract In wide-field super-resolution microscopy, investigating the nanoscale structure of cellular processes, and resolving fast dynamics and morphological changes in cells requires algorithms capable of working with a high-density of emissive fluorophores. Current deconvolution algorithms estimate fluorophore density by using representations of the signal that promote sparsity of the super-resolution images via an L1-norm penalty. This penalty imposes a restriction on the sum of absolute values of the estimates of emitter brightness. By implementing an L0-norm penalty – on the number of fluorophores rather than on their overall brightness – we present a penalized regression approach that can work at high-density and allows fast super-resolution imaging. We validated our approach on simulated images with densities up to 15 emitters per μm-2 and investigated total internal reflection fluorescence (TIRF) data of mitochondria in a HEK293-T cell labeled with DAKAP-Dronpa. We demonstrated super-resolution imaging of the dynamics with a resolution down to 55 nm and a 0.5 s time sampling.

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 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.

Applications of nanostructures in wide-field, label-free super resolution microscopy

2018 ◽  
Vol 27 (11) ◽  
pp. 118704 ◽  
Author(s):  
Xiaowei Liu ◽  
Chao Meng ◽  
Xuechu Xu ◽  
Mingwei Tang ◽  
Chenlei Pang ◽  
...  
Keyword(s):  
Super Resolution ◽  
Wide Field ◽  
Label Free ◽  
Super Resolution Microscopy

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 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.

DAOSTORM: an algorithm for high- density super-resolution microscopy

2011 ◽  
Vol 8 (4) ◽  
pp. 279-280 ◽  
Author(s):  
Seamus J Holden ◽  
Stephan Uphoff ◽  
Achillefs N Kapanidis
Keyword(s):  
Super Resolution ◽  
High Density ◽  
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.

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