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 Spatial filter and its application in three-dimensional single molecule localization microscopy

2020 ◽  
Vol 8 (2) ◽  
pp. 025008
Author(s):  
Xiaoming Fan ◽  
Johnny Hendriks ◽  
Maddalena Comini ◽  
Alexandros Katranidis ◽  
Georg Büldt ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Spatial Filter ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

2018 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Double Helix ◽  
Super Resolution ◽  
Large Set ◽  
Holistic View ◽  
Localization Microscopy ◽  
Fight Club ◽  
Community Effort ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

ABSTRACTWith the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities – 2D, astigmatic 3D, biplane 3D, and double helix 3D – and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

scholarly journals Three-dimensional biplane spectroscopic single-molecule localization microscopy: erratum

Optica ◽  
2019 ◽  
Vol 6 (10) ◽  
pp. 1374
Author(s):  
Ki-Hee Song ◽  
Yang Zhang ◽  
Gaoxiang Wang ◽  
Cheng Sun ◽  
Hao F. Zhang
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Photon count estimation in single-molecule localization microscopy

2018 ◽  
Author(s):  
Rasmus Ø. Thorsen ◽  
Christiaan N. Hulleman ◽  
Mathias Hammer ◽  
David Grünwald ◽  
Sjoerd Stallinga ◽  
...  
Keyword(s):  
Single Molecule ◽  
Axial Position ◽  
Optical Aberrations ◽  
Intensity Estimation ◽  
Localization Microscopy ◽  
Photon Count ◽  
Point Spread ◽  
Spread Function ◽  
Gaussian Point ◽  
Single Molecule Localization Microscopy

Recently, Franke, Sauer and van de Linde1 introduced a way to estimate the axial position of single-molecules (TRABI). To this end, they compared the detected photon count from a temporal radial-aperture-based intensity estimation to the estimated count from Gaussian point-spread function (PSF) fitting to the data. Empirically they found this photometric ratio to be around 0.7-0.8 close to focus and decreasing away from it. Here, we explain this reported but unexplained discrepancy and furthermore show that the photometric ratio as indicator for axial position is susceptible even to typical optical aberrations.

scholarly journals Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells

2020 ◽  
Vol 6 (16) ◽  
pp. eaay8271 ◽  
Author(s):  
Simao Coelho ◽  
Jongho Baek ◽  
Matthew S. Graus ◽  
James M. Halstead ◽  
Philip R. Nicovich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Distance Measurements ◽  
Intact Cells ◽  
Localization Microscopy ◽  
Direct Distance ◽  
Time Drift ◽  
Localization Precision ◽  
20 Nm ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

scholarly journals Three dimensional drift control at nano-scale in single molecule localization microscopy

2020 ◽  
Vol 28 (22) ◽  
pp. 32750
Author(s):  
Xiaoming Fan ◽  
Thomas Gensch ◽  
Georg Büldt ◽  
Yuanheng Zhang ◽  
Zulipali Musha ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Localization Microscopy ◽  
Nano Scale ◽  
Single Molecule Localization Microscopy ◽  
Drift Control
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