Spatial filter and its application in three-dimensional 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.

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

Optics Express ◽

10.1364/oe.404123 ◽

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

10.1101/2020.04.15.043182 ◽

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.

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Symmetrically-dispersed spectroscopic single-molecule localization microscopy

10.1101/2019.12.14.876557 ◽

2019 ◽

Author(s):

K. Song ◽

Y. Zhang ◽

B. Brenner ◽

C. Sun ◽

H. F. Zhang

Keyword(s):

Spectral Analysis ◽

Single Molecule ◽

Particle Tracking ◽

Three Dimensional ◽

Spatial Localization ◽

Single Particle Tracking ◽

Localization Microscopy ◽

First Time ◽

Spectral Channels ◽

Single Molecule Localization Microscopy

AbstractSpectroscopic single-molecule localization microscopy (sSMLM) achieved simultaneously imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from reduced photon budget because of dividing photons from individual stochastic emission into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically-dispersed sSMLM or SDsSMLM to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Comparing with existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multi-color imaging in fixed cells and three-dimensional single-particle tracking using SDsSMLM.

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Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

10.1101/568279 ◽

2019 ◽

Author(s):

Martin Pauli ◽

Mila M. Paul ◽

Sven Proppert ◽

Marzieh Sharifi ◽

Felix Repp ◽

...

Keyword(s):

Single Molecule ◽

Mossy Fiber ◽

Three Dimensional ◽

Brain Regions ◽

Molecular Organization ◽

Localization Microscopy ◽

Stochastic Optical Reconstruction Microscopy ◽

Optical Reconstruction ◽

Axial Scanning ◽

Single Molecule Localization Microscopy

ABSTRACTRevealing the molecular organization of anatomically precisely defined brain regions is necessary for the refined understanding of synaptic plasticity. Although, three-dimensional (3D) single-molecule localization microscopy can provide the required molecular resolution, single-molecule imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for aberration-free volumetricdirectstochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enables 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

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Three-dimensional single-molecule localization microscopy imaging based on compressed sensing

Chinese Optics ◽

10.37188/co.2020-0003 ◽

2020 ◽

Vol 13 (5) ◽

pp. 1065-1074

Author(s):

ZHANG Sai-wen ◽

◽

LIN Dan-ying ◽

YU Bin ◽

LENG Xiao-ling ◽

...

Keyword(s):

Compressed Sensing ◽

Single Molecule ◽

Three Dimensional ◽

Microscopy Imaging ◽

Localization Microscopy ◽

Single Molecule Localization Microscopy

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Interferometric three-dimensional single molecule localization microscopy using a single high-numerical-aperture objective

Applied Optics ◽

10.1364/ao.53.007415 ◽

2014 ◽

Vol 53 (31) ◽

pp. 7415 ◽

Cited By ~ 1

Author(s):

P. Zhang ◽

P. M. Goodwin ◽

J. H. Werner

Keyword(s):

Single Molecule ◽

Three Dimensional ◽

Numerical Aperture ◽

Localization Microscopy ◽

High Numerical Aperture ◽

Single Molecule Localization Microscopy

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Hochaufgelöste Visualisierung einzelner Moleküle auf ganzen Zellen

BIOspektrum ◽

10.1007/s12268-020-1501-4 ◽

2020 ◽

Vol 26 (7) ◽

pp. 735-738

Author(s):

Jan Schlegel ◽

Markus Sauer

Keyword(s):

Single Molecule ◽

Three Dimensional ◽

Light Sheet ◽

Localization Microscopy ◽

Adhesion Receptor ◽

Whole Cells ◽

Analysis Techniques ◽

Complex Interactions ◽

Different Parts ◽

Single Molecule Localization Microscopy

AbstractBiological systems are dynamic and three-dimensional but many techniques allow only static and two-dimensional observation of cells. We used three-dimensional (3D) lattice light-sheet single-molecule localization microscopy (dSTORM) to investigate the complex interactions and distribution of single molecules in the plasma membrane of whole cells. Different receptor densities of the adhesion receptor CD56 at different parts of the cell highlight the importance and need of three-dimensional observation and analysis techniques.

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