scholarly journals Fast, robust and precise 3D localization for arbitrary point spread functions

2017 ◽  
Author(s):  
Yiming Li ◽  
Markus Mund ◽  
Philipp Hoess ◽  
Ulf Matti ◽  
Bianca Nijmeijer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Arbitrary Point ◽  
Experimental Point ◽  
High Quality ◽  
Point Spread Functions ◽  
3D Localization ◽  
Point Spread ◽  
Minimum Uncertainty

AbstractWe present a fitter for 3D single-molecule localization of arbitrary, experimental point spread functions (PSFs) that reaches minimum uncertainty for EMCCD and sCMOS cameras, and achieves more than 105 fits/s. We provide tools to robustly model experimental PSFs and correct for depth induced aberrations, which allowed us to achieve an unprecedented 3D resolution with engineered astigmatic PSFs, and acquire high quality 3D superresolution images even on standard microscopes without 3D optics.

scholarly journals Real-time 3D single-molecule localization using experimental point spread functions

2018 ◽  
Vol 15 (5) ◽  
pp. 367-369 ◽  
Author(s):  
Yiming Li ◽  
Markus Mund ◽  
Philipp Hoess ◽  
Joran Deschamps ◽  
Ulf Matti ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Experimental Point ◽  
Point Spread Functions ◽  
Point Spread

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 Analyzing engineered point spread functions using phasor-based single-molecule localization microscopy

2020 ◽  
Author(s):  
Koen J.A. Martens ◽  
Abbas Jabermoradi ◽  
Suyeon Yang ◽  
Johannes Hohlbein
Keyword(s):  
Saddle Point ◽  
Single Molecule ◽  
Software Package ◽  
Double Helix ◽  
Three Dimensional ◽  
Fourier Plane ◽  
Point Spread Functions ◽  
Localization Microscopy ◽  
Point Spread ◽  
Single Molecule Localization Microscopy

The point spread function (PSF) of single molecule emitters can be engineered in the Fourier plane to encode three-dimensional localization information, creating double-helix, saddle-point or tetra-pod PSFs. Here, we describe and assess adaptations of the phasor-based single-molecule localization microscopy (pSMLM) algorithm to localize single molecules using these PSFs with sub-pixel accuracy. For double-helix, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations, while for saddle-point or tetra-pod, a novel phasor-based deconvolution approach is used. The pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores. pSMLM substantially improves the localization rate by a factor of 2 - 4x on a standard CPU, with 1-1.5·104 (double-helix) or 2.5·105 (saddle-point/tetra-pod) localizations/second.

scholarly journals Observation of live chromatin dynamics in cells via 3D localization microscopy using Tetrapod point spread functions

2017 ◽  
Vol 8 (12) ◽  
pp. 5735 ◽  
Author(s):  
Yoav Shechtman ◽  
Anna-Karin Gustavsson ◽  
Petar N. Petrov ◽  
Elisa Dultz ◽  
Maurice Y. Lee ◽  
...  
Keyword(s):  
Point Spread Functions ◽  
Chromatin Dynamics ◽  
Localization Microscopy ◽  
3D Localization ◽  
Point Spread ◽  
In Cells
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