Dual responsive specifically labelled carbogenic fluorescent nanodots for super resolution and electron microscopy

Nanoscale ◽  
2019 ◽  
Vol 11 (14) ◽  
pp. 6561-6565 ◽  
Author(s):  
Navneet. C. Verma ◽  
Chethana Rao ◽  
Ashutosh Singh ◽  
Neha Garg ◽  
Chayan K. Nandi
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Dual Responsive ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Transmission Electron ◽  
Optical Reconstruction

We introduce an orange emissive fluorescent nanodot for successful single molecule stochastic optical reconstruction microscopy (STORM), super resolution radial fluctuation (SRRF) microscopy and transmission electron microscopy (TEM).

scholarly journals Precision of fiducial marker alignment for correlative super‐resolution fluorescence and transmission electron microscopy

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Adeeba Fathima ◽  
César Augusto Quintana-Cataño ◽  
Christoph Heintze ◽  
Michael Schlierf
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Specific Protein ◽  
Fiducial Markers ◽  
Transmission Electron ◽  
Microscopy Techniques

AbstractRecent advances in microscopy techniques enabled nanoscale discoveries in biology. In particular, electron microscopy reveals important cellular structures with nanometer resolution, yet it is hard, and sometimes impossible to resolve specific protein localizations. Super-resolution fluorescence microscopy techniques developed over the recent years allow for protein-specific localization with ~ 20 nm precision are overcoming this limitation, yet it remains challenging to place those in cells without a reference frame. Correlative light and electron microscopy (CLEM) approaches have been developed to place the fluorescence image in the context of a cellular structure. However, combining imaging methods such as super resolution microscopy and transmission electron microscopy necessitates a correlation using fiducial markers to locate the fluorescence on the structures visible in electron microscopy, with a measurable precision. Here, we investigated different fiducial markers for super-resolution CLEM (sCLEM) by evaluating their shape, intensity, stability and compatibility with photoactivatable fluorescent proteins as well as the electron density. We further carefully determined limitations of correlation accuracy. We found that spectrally-shifted FluoSpheres are well suited as fiducial markers for correlating single-molecule localization microscopy with transmission electron microscopy.

scholarly journals Stop-Frame Filming and Discovery of Reactions at the Single-Molecule Level by Transmission Electron Microscopy

ACS Nano ◽  
2017 ◽  
Vol 11 (3) ◽  
pp. 2509-2520 ◽  
Author(s):  
Thomas W. Chamberlain ◽  
Johannes Biskupek ◽  
Stephen T. Skowron ◽  
Alexander V. Markevich ◽  
Simon Kurasch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Single Molecule ◽  
Single Molecule Level ◽  
Transmission Electron

scholarly journals Resolution enhancement of transmission electron microscopy by super-resolution radial fluctuations

2020 ◽  
Vol 116 (4) ◽  
pp. 044105
Author(s):  
Y. Zhang ◽  
S. Rouvimov ◽  
X. Yuan ◽  
K. Gonzalez-Serrano ◽  
A. C. Seabaugh ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Resolution Enhancement ◽  
Super Resolution ◽  
Transmission Electron

scholarly journals Tetra-gel enables superior accuracy in combined super-resolution imaging and expansion microscopy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hsuan Lee ◽  
Chih-Chieh Yu ◽  
Edward S. Boyden ◽  
Xiaowei Zhuang ◽  
Pallav Kosuri
Keyword(s):  
Standard Deviation ◽  
Single Molecule ◽  
Super Resolution ◽  
Structural Features ◽  
Chain Growth ◽  
Radical Chain ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Direct Measurements ◽  
Optical Reconstruction ◽  
Structural Preservation

AbstractThe accuracy of expansion microscopy (ExM) depends on the structural preservation of samples embedded in a hydrogel. However, it has been unknown to what extent gel embedding alters the molecular positions of individual labeled sites. Here, we quantified the accuracy of gel embedding by using stochastic optical reconstruction microscopy (STORM) to image DNA origami with well-defined structures. We found that embedding in hydrogels based on polyacrylamide, the most widely used chemistry in ExM, resulted in random displacements of labeled sites with a standard deviation of ~ 16 nm. In contrast, we found that embedding in tetra-gel, a hydrogel that does not depend on free-radical chain-growth polymerization, preserved labeled sites with a standard deviation of less than 5 nm. By combining tetra-gel ExM with STORM, we were able to resolve 11-nm structural features without the loss in accuracy seen with polyacrylamide gels. Our study thus provides direct measurements of the single-molecule distortions resulting from hydrogel embedding, and presents a way to improve super-resolution microscopy through combination with tetra-gel ExM.

scholarly journals About samples, giving examples: Optimized Single Molecule Localization Microscopy

2019 ◽  
Author(s):  
Angélique Jimenez ◽  
Karoline Friedl ◽  
Christophe Leterrier
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Full Potential ◽  
Coated Pits ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Nanoscale Topography ◽  
Biological Discovery ◽  
Optical Reconstruction ◽  
Single Molecule Localization Microscopy

AbstractSuper-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

scholarly journals Volumetric super-resolution imaging by serial ultrasectioning and STochastic Optical Reconstruction Microscopy (STORM) in neural tissue

2021 ◽  
Author(s):  
Tarlan Vatan ◽  
Jacqueline A. Minehart ◽  
Chenghang Zhang ◽  
Vatsal Agarwal ◽  
Jerry Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Neural Tissue ◽  
Imaging Data ◽  
Whole Cells ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Tissue Scattering ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Chemical Synapses

Abstract Here we present a protocol for collecting large-volume, four-color, single-molecule localization imaging data from neural tissue. We have applied this technique to map the location and identities of chemical synapses across whole cells in mouse retinae. Our sample preparation approach improves 3D STORM image quality by reducing tissue scattering, photobleaching, and optical distortions associated with deep imaging. This approach can be extended for use on other tissue types enabling life scientists to perform volumetric super-resolution imaging in diverse biological models. For a detailed application of this protocol, please refer to Sigal et al., 2015.

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