scholarly journals Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

2018 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Double Helix ◽  
Super Resolution ◽  
Large Set ◽  
Holistic View ◽  
Localization Microscopy ◽  
Fight Club ◽  
Community Effort ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

ABSTRACTWith the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities – 2D, astigmatic 3D, biplane 3D, and double helix 3D – and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

scholarly journals Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

2019 ◽  
Vol 16 (5) ◽  
pp. 387-395 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Fight Club ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

scholarly journals Publisher Correction: Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

2019 ◽  
Vol 16 (6) ◽  
pp. 561-561 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Fight Club ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

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 Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

2021 ◽  
Author(s):  
Nicolas Lardon ◽  
Lu Wang ◽  
Aline Tschanz ◽  
Philipp Hoess ◽  
Mai Tran ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Range ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Important Class ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

scholarly journals Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS Cameras

2017 ◽  
Author(s):  
Hazen P. Babcock
Keyword(s):  
Open Source ◽  
Single Molecule ◽  
Open Source Software ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Industrial Grade ◽  
Spectrally Resolved ◽  
Imaging Plane ◽  
Single Molecule Localization Microscopy

ABSTRACTIn this work we explore the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that the performance of these cameras in single imaging plane SMLM applications is comparable to much more expensive scientific CMOS (sCMOS) cameras. We show that these cameras can be used in more demanding biplane, multiplane and spectrally resolved SMLM applications. The 10-40× reduction in camera cost makes it practical to build SMLM setups with 4 or more cameras. In addition we provide open-source software for simultaneously controlling multiple CMOS cameras and for the reduction of the movies that are acquired to super-resolution images.

A literature review of localization methods and emergence of deep learning in single-molecule localization microscopy

2020 ◽  
Author(s):  
Anish Mukherjee
Keyword(s):  
Deep Learning ◽  
Single Molecule ◽  
Super Resolution ◽  
Point Sources ◽  
Learning Approaches ◽  
Localization Algorithm ◽  
Localization Microscopy ◽  
Trade Offs ◽  
Emitter Localization ◽  
Single Molecule Localization Microscopy

The quality of super-resolution images largely depends on the performance of the emitter localization algorithm used to localize point sources. In this article, an overview of the various techniques which are used to localize point sources in single-molecule localization microscopy are discussed and their performances are compared. This overview can help readers to select a localization technique for their application. Also, an overview is presented about the emergence of deep learning methods that are becoming popular in various stages of single-molecule localization microscopy. The state of the art deep learning approaches are compared to the traditional approaches and the trade-offs of selecting an algorithm for localization are discussed.

scholarly journals Photoactivation of silicon rhodamines via a light-induced protonation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Michelle S. Frei ◽  
Philipp Hoess ◽  
Marko Lampe ◽  
Bianca Nijmeijer ◽  
Moritz Kueblbeck ◽  
...  
Keyword(s):  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Super Resolution ◽  
Spectroscopic Properties ◽  
Single Particle Tracking ◽  
Live Cell ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Abstract Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

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