Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization and light sheet illumination

AbstractOver the last decades, super-resolution techniques have revolutionized the field of fluorescence microscopy. Among them, interferometric or 4Pi microscopy methods exhibit supreme resolving power in the axial dimension. Combining with single-molecule detection/localization and adaptive optics, iPALM/4PiSMS/W-4PiSMSN allowed 10-15 nm isotropic 3D resolution throughout the whole cell. However, further improving the achieved 3D resolution poses significantly challenges which, in part, is blocked by the complexity of single-molecule emission pattern generated by these systems rendering a large portion of information carrying photons unusable. Here we introduce a localization algorithm that achieves the theoretical information limit for 4Pi based single-molecule switching nanoscopy (4Pi-SMSN), and demonstrates improvements in resolution, accuracy as well as applicability comparing with the state of art 4Pi-SMSN methods. Further, with a novel 4Pi-compatible light-sheet illumination reducing the fluorescence background by >5-fold, we demonstrated the new system enables further improvement in the achievable resolution of 4Pi/interferometric single-molecule imaging systems.

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Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization

Communications Biology ◽

10.1038/s42003-020-0908-2 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 4

Author(s):

Sheng Liu ◽

Fang Huang

Keyword(s):

Adaptive Optics ◽

Single Molecule ◽

Super Resolution ◽

Resolving Power ◽

Localization Algorithm ◽

Whole Cells ◽

4Pi Microscopy ◽

Axial Dimension ◽

Localization Precision ◽

Single Molecule Localization Microscopy

AbstractOver the last decades, super-resolution techniques have revolutionized the field of fluorescence microscopy. Among them, interferometric or 4Pi microscopy methods exhibit supreme resolving power in the axial dimension. Combined with single-molecule detection/localization and adaptive optics, current 4Pi microscopy methods enabled 10–15 nm isotropic 3D resolution throughout whole cells. However, further improving the achieved 3D resolution poses challenges arising from the complexity of single-molecule emission patterns generated by these coherent single-molecule imaging systems. These complex emission patterns render a large portion of information carrying photons unusable. Here, we introduce a localization algorithm that achieves the theoretical precision limit for a 4Pi based single-molecule switching nanoscopy (4Pi-SMSN) system, and demonstrate improvements in localization precision, accuracy as well as stability comparing with state-of-the-art 4Pi-SMSN methods.

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A literature review of localization methods and emergence of deep learning in single-molecule localization microscopy

10.31219/osf.io/y62rq ◽

2020 ◽

Author(s):

Anish Mukherjee

Keyword(s):

Deep Learning ◽

Single Molecule ◽

Super Resolution ◽

Point Sources ◽

Learning Approaches ◽

Localization Algorithm ◽

Localization Microscopy ◽

Trade Offs ◽

Emitter Localization ◽

Single Molecule Localization Microscopy

The quality of super-resolution images largely depends on the performance of the emitter localization algorithm used to localize point sources. In this article, an overview of the various techniques which are used to localize point sources in single-molecule localization microscopy are discussed and their performances are compared. This overview can help readers to select a localization technique for their application. Also, an overview is presented about the emergence of deep learning methods that are becoming popular in various stages of single-molecule localization microscopy. The state of the art deep learning approaches are compared to the traditional approaches and the trade-offs of selecting an algorithm for localization are discussed.

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Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines

10.1101/083402 ◽

2016 ◽

Cited By ~ 2

Author(s):

Hazen P. Babcock ◽

Xiaowei Zhuang

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Computation Time ◽

Cubic Splines ◽

Localization Algorithm ◽

Localization Microscopy ◽

Analytical Functions ◽

Open Source Software Package ◽

Microscopy Data ◽

Single Molecule Localization Microscopy

AbstractThe resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2-3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed.

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Oblique plane single-molecule localization microscopy for thick samples

10.1101/289686 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jeongmin Kim ◽

Michal Wojcik ◽

Yuan Wang ◽

Ke Xu ◽

Xiang Zhang

Keyword(s):

Fluorescence Detection ◽

Single Molecule ◽

Tissue Sample ◽

Super Resolution ◽

Light Sheet ◽

Localization Microscopy ◽

Isotropic Resolution ◽

Fluorescent Beads ◽

Imaging Depth ◽

Single Molecule Localization Microscopy

We introduce single-molecule oblique plane microscopy (obSTORM) to directly image oblique sections of thick samples into depth without lengthy axial stack acquisition. Using oblique light-sheet illumination and oblique fluorescence detection, obSTORM offers uniform super-resolution throughout imaging depth in diverse biological specimens from cells to tissues. In particular, we demonstrate an isotropic resolution of ∼51 nm over a depth of 32 μm for a tissue sample, and comparable resolution over a depth of 100 μm using fluorescent beads.

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Quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy

10.1101/721837 ◽

2019 ◽

Author(s):

Hesam Mazidi ◽

Tianben Ding ◽

Arye Nehorai ◽

Matthew D. Lew

Keyword(s):

Single Molecule ◽

Computational Models ◽

Amyloid Fibrils ◽

Imaging System ◽

Simulated Data ◽

Super Resolution ◽

Ground Truth ◽

Localization Algorithm ◽

Reconstruction Algorithms ◽

Imaging Data

The resolution and accuracy of single-molecule localization micro-scopes (SMLMs) are routinely benchmarked using simulated data, calibration “rulers,” or comparisons to secondary imaging modalities. However, these methods cannot quantify the nanoscale accuracy of an arbitrary SMLM dataset. Here, we show that by computing localization stability under a well-chosen perturbation with accurate knowledge of the imaging system, we can robustly measure the confidence of individual localizations without ground-truth knowledge of the sample. We demonstrate that our method, termed Wasserstein-induced flux (WIF), measures the accuracy of various reconstruction algorithms directly on experimental 2D and 3D data of microtubules and amyloid fibrils. We further show that WIF confidences can be used to evaluate the mismatch between computational models and imaging data, enhance the accuracy and resolution of recon-structed structures, and discover hidden molecular heterogeneities. As a computational methodology, WIF is broadly applicable to any SMLM dataset, imaging system, and localization algorithm.

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Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): an algorithm for MHz localization rates using standard CPUs

10.1101/191957 ◽

2017 ◽

Cited By ~ 1

Author(s):

Koen J.A. Martens ◽

Arjen N. Bader ◽

Sander Baas ◽

Bernd Rieger ◽

Johannes Hohlbein

Keyword(s):

Single Molecule ◽

Iterative Algorithms ◽

Region Of Interest ◽

Depth Information ◽

Localization Algorithm ◽

Two Phase ◽

Model Free ◽

Spread Function ◽

2D And 3D ◽

Single Molecule Localization Microscopy

AbstractWe present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier coefficients in both x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

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Enabling single-molecule localization microscopy in turbid food emulsions

10.1101/2021.03.03.433739 ◽

2021 ◽

Author(s):

Abbas Jabermoradi ◽

Suyeon Yang ◽

Martijn Gobes ◽

John P.M. van Duynhoven ◽

Johannes Hohlbein

Keyword(s):

Adaptive Optics ◽

Single Molecule ◽

Optical Resolution ◽

Three Dimensions ◽

Water Interface ◽

Water Emulsion ◽

Food Emulsions ◽

Localization Microscopy ◽

Oil Water ◽

Single Molecule Localization Microscopy

Turbidity poses a major challenge for the microscopic characterization of many food systems. In these systems, local mismatches in refractive indices can cause reflection, absorption and scattering of incoming as well as outgoing light leading to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the achievable optical resolution, we combined adaptive optics (AO) with single-molecule localization microscopy (SMLM). Building on our previously published open hardware microscopy framework, the miCube, we first added a deformable mirror to the detection path. This element enables both the compensation of aberrations directly from single-molecule data and, by further modulating the emission wavefront, the introduction of various point spread functions (PSFs) to enable SMLM in three dimensions. We further added a top hat beam shaper to the excitation path to obtain an even illumination profile across the field of view (FOV). As a model system for a non-transparent food colloid in which imaging in depth is challenging, we designed an oil-in-water emulsion in which phosvitin, a ferric ion binding protein present in from egg yolk, resides at the oil water interface. We targeted phosvitin with fluorescently labelled primary antibodies and used PSF engineering to obtain 2D and 3D images of phosvitin covered oil droplets with sub 100 nm resolution. Droplets with radii as low as 200 nm can be discerned, which is beyond the range of conventional confocal light microscopy. Our data indicated that in the model emulsion phosvitin is homogeneously distributed at the oil-water interface. With the possibility to obtain super-resolved images in depth of nontransparent colloids, our work paves the way for localizing biomacromolecules at colloidal interfaces in heterogeneous food emulsions.

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