scholarly journals Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors

2018 ◽  
Author(s):  
Tomáš Lukeš ◽  
Jakub Pospíšil ◽  
Karel Fliegel ◽  
Theo Lasser ◽  
Guy M. Hagen
Keyword(s):  
Data Analysis ◽  
Growth Factor ◽  
Single Molecule ◽  
Super Resolution ◽  
Growth Factor Receptors ◽  
Data Sets ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Resolution Single ◽  
Single Molecule Localization Microscopy

BackgroundSuper-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared to organic dyes which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms.FindingsFour complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM data sets using a different method: super-resolution optical fluctuation imaging (SOFI). The two modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes.ConclusionThis dataset has potential for extensive reuse. Complete raw data from SMLM experiments has typically not been published. The YFP data exhibits low signal to noise ratios, making data analysis a challenge. These data sets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

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 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.

scholarly journals ShareLoc – an open platform for sharing localization microscopy data

2021 ◽  
Author(s):  
Jiachuan Bai ◽  
Wei Ouyang ◽  
Manish Kumar Singh ◽  
Christophe Leterrier ◽  
Paul Barthelemy ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Data Sets ◽  
Localization Microscopy ◽  
Open Platform ◽  
Machine Learning Methods ◽  
Microscopy Data ◽  
Analytical Tools ◽  
Existing Data ◽  
Single Molecule Localization Microscopy

Novel insights and more powerful analytical tools can emerge from the reanalysis of existing data sets, especially via machine learning methods. Despite the widespread use of single molecule localization microscopy (SMLM) for super-resolution bioimaging, the underlying data are often not publicly accessible. We developed ShareLoc (https://shareloc.xyz), an open platform designed to enable sharing, easy visualization and reanalysis of SMLM data. We discuss its features and show how data sharing can improve the performance and robustness of SMLM image reconstruction by deep learning.

scholarly journals Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single Molecule Localization Microscopy

2021 ◽  
Author(s):  
Siddharth Matikonda ◽  
Dominic Helmerich ◽  
Mara Meub ◽  
Gerti Beliu ◽  
Philip Kollmannsberger ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Computational Analysis ◽  
Super Resolution ◽  
Underlying Mechanism ◽  
New Approach ◽  
Localization Microscopy ◽  
Order Of Magnitude ◽  
Resolution Single ◽  
Single Molecule Localization Microscopy

<p>The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multi-step sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.</p>

scholarly journals Visualizing multi-protein patterns at the synapse of neuronal tissue with DNA-assisted single-molecule localization microscopy

2021 ◽  
Author(s):  
Kaarjel K. Narayanasamy ◽  
Aleksandar Stojic ◽  
Yunqing Li ◽  
Steffen Sass ◽  
Marina Hesse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Super Resolution ◽  
Protein Patterns ◽  
Multiple Targets ◽  
Localization Microscopy ◽  
Neuronal Tissue ◽  
Tissue Sections ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractThe development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy. Using antibodies labeled with short DNA oligonucleotides, multiple targets are visualized successively by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. The structural integrity of the tissue is maintained owing to only a single labelling step during sample preparation. Multiple targets are imaged using a single laser wavelength, minimizing chromatic aberration. This method proved robust for multi-target imaging in semi-thin tissue sections, paving the way towards structural cell biology with single-molecule super-resolution microscopy.

scholarly journals DNA-Based Super-Resolution Microscopy: DNA-PAINT

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 621 ◽  
Author(s):  
Daniel Nieves ◽  
Katharina Gaus ◽  
Matthew Baker
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Cutting Edge ◽  
Cellular Proteins ◽  
Localization Microscopy ◽  
Nanoscale Topography ◽  
History Of ◽  
Super Resolution Microscopy ◽  
Fluorescent Ligand ◽  
Single Molecule Localization Microscopy

Super-resolution microscopies, such as single molecule localization microscopy (SMLM), allow the visualization of biomolecules at the nanoscale. The requirement to observe molecules multiple times during an acquisition has pushed the field to explore methods that allow the binding of a fluorophore to a target. This binding is then used to build an image via points accumulation for imaging nanoscale topography (PAINT), which relies on the stochastic binding of a fluorescent ligand instead of the stochastic photo-activation of a permanently bound fluorophore. Recently, systems that use DNA to achieve repeated, transient binding for PAINT imaging have become the cutting edge in SMLM. Here, we review the history of PAINT imaging, with a particular focus on the development of DNA-PAINT. We outline the different variations of DNA-PAINT and their applications for imaging of both DNA origamis and cellular proteins via SMLM. Finally, we reflect on the current challenges for DNA-PAINT imaging going forward.

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