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 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 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 3D single-molecule super-resolution microscopy with a tilted light sheet

2017 ◽  
Author(s):  
Anna-Karin Gustavsson ◽  
Petar N. Petrov ◽  
Maurice Y. Lee ◽  
Yoav Shechtman ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Double Helix ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Light Sheet ◽  
Point Spread Functions ◽  
Light Sheet Microscopy ◽  
Point Spread ◽  
Resolution Imaging

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSFs for fiducial bead tracking and live axial drift correction.

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