scholarly journals Combining 3D single molecule localization strategies for reproducible bioimaging

2018 ◽  
Author(s):  
Clément Cabriel ◽  
Nicolas Bourg ◽  
Pierre Jouchet ◽  
Guillaume Dupuis ◽  
Christophe Leterrier ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
3D Localization ◽  
Point Spread ◽  
Spread Function ◽  
Multicolor Imaging ◽  
Localization Precision ◽  
Different Depths ◽  
Super Resolution Microscopy ◽  
Living Bacteria

We developed a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1 μm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently to provide a weakly anisotropic 3D resolution over the imaging range. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several-μm range by direct merging of multiple acquisitions at different depths.

scholarly journals 3D Single-Molecule Super-Resolution Fluorescence Microscopy with the Corkscrew Point Spread Function

2016 ◽  
Vol 110 (3) ◽  
pp. 176a
Author(s):  
Maurice Lee ◽  
Matthew Lew ◽  
Alex von Diezmann ◽  
Lucien Weiss ◽  
Yoav Shechtman ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Point Spread Function ◽  
Super Resolution ◽  
Point Spread ◽  
Spread Function

The changing point-spread function: single-molecule-based super-resolution imaging

2014 ◽  
Vol 141 (6) ◽  
pp. 577-585 ◽  
Author(s):  
Mathew H. Horrocks ◽  
Matthieu Palayret ◽  
David Klenerman ◽  
Steven F. Lee
Keyword(s):  
Single Molecule ◽  
Point Spread Function ◽  
Super Resolution ◽  
Point Spread ◽  
Spread Function ◽  
Resolution Imaging

scholarly journals Minimizing Structural Bias in Single-Molecule Super-Resolution Microscopy

2018 ◽  
Author(s):  
Hesam Mazidi ◽  
Jin Lu ◽  
Arye Nehorai ◽  
Matthew D. Lew
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Estimation Algorithm ◽  
Image Resolution ◽  
Spread Function ◽  
Localization Errors ◽  
Imaging Artifacts ◽  
Detection And Localization ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

ABSTRACTSingle-molecule localization microscopy (SMLM) depends on sequential detection and localization of individual molecular blinking events. Due to the stochasticity of single-molecule blinking and the desire to improve SMLM’s temporal resolution, algorithms capable of analyzing frames with a high density (HD) of active molecules, or molecules whose images overlap, are a prerequisite for accurate location measurements. Thus far, HD algorithms are evaluated using scalar metrics, such as root-mean-square error, that fail to quantify the structure of errors caused by the structure of the sample. Here, we show that the spatial distribution of localization errors within super-resolved images of biological structures are vectorial in nature, leading to systematic structural biases that severely degrade image resolution. We further demonstrate that the shape of the microscope’s point-spread function (PSF) fundamentally affects the characteristics of imaging artifacts. We built a Robust Statistical Estimation algorithm (RoSE) to minimize these biases for arbitrary structures and PSFs. RoSE accomplishes this minimization by estimating the likelihood of blinking events to localize molecules more accurately and eliminate false localizations. Using RoSE, we measure the distance between crossing microtubules, quantify the morphology of and separation between vesicles, and obtain robust recovery using diverse 3D PSFs with unmatched accuracy compared to state-of-the-art algorithms.

scholarly journals Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jagadish Sankaran ◽  
Harikrushnan Balasubramanian ◽  
Wai Hoh Tang ◽  
Xue Wen Ng ◽  
Adrian Röllin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Temporal Dynamics ◽  
Growth Factor Receptor ◽  
Super Resolution ◽  
Actin Binding ◽  
Biological Knowledge ◽  
Data Set ◽  
Epidermal Growth ◽  
Molecular Brightness ◽  
Super Resolution Microscopy

AbstractSuper-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.

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.

Sign in / Sign up

Export Citation Format

Share Document