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

2020 ◽  
Vol 3 (1) ◽  
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.

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

2019 ◽  
Author(s):  
Sheng Liu ◽  
Fang Huang
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Resolving Power ◽  
Localization Algorithm ◽  
Emission Pattern ◽  
4Pi Microscopy ◽  
Axial Dimension ◽  
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. 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.

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

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.

scholarly journals Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines

2016 ◽  
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.

scholarly journals Restoring single-molecule localizations with wavefront sensing adaptive optics for deep-tissue super-resolution imaging

2021 ◽  
Author(s):  
Sanghyeon Park ◽  
Yonghyeon Jo ◽  
Minsu Kang ◽  
Jin Hee Hong ◽  
Sangyoon Ko ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Fluorescence Emission ◽  
Super Resolution ◽  
Light Modulator ◽  
Application Range ◽  
Wavefront Distortion ◽  
Resolution Imaging ◽  
Localization Precision ◽  
Major Factors

Specimen-induced aberration has been one of the major factors limiting the imaging depth in single-molecule localization microscopy (SMLM). In this study, we measured the wavefront of intrinsic reflectance signal at the fluorescence emission wavelength to construct a time-gated reflection matrix and find complex tissue aberration without resorting to fluorescence detection. Physically correcting the identified aberration via wavefront shaping with a liquid-crystal spatial light modulator (SLM) enables super-resolution imaging even when the aberration is too severe for initiating localization processes. We demonstrate the correction of complex tissue aberration, the root-mean-square (RMS) wavefront distortion of which is more than twice the 1 rad limit presented in previous studies; this leads to the recovery of single molecules by 77 times increased localization number. We visualised dendritic spines in mouse brain tissues and early myelination processes in a whole zebrafish at up to 102 μm depth with 28-39 nm localization precision. The proposed approach can expand the application range of SMLM to thick samples that cause the loss of localization points owing to severe aberration.

scholarly journals Parameter-free rendering of single-molecule localization microscopy data for parameter-free resolution estimation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Adrien C. Descloux ◽  
Kristin S. Grußmayer ◽  
Aleksandra Radenovic
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Free Resolution ◽  
Temporal Separation ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Localization Precision ◽  
Microscopy Data ◽  
Resolution Estimation ◽  
Single Molecule Localization Microscopy

AbstractLocalization microscopy is a super-resolution imaging technique that relies on the spatial and temporal separation of blinking fluorescent emitters. These blinking events can be individually localized with a precision significantly smaller than the classical diffraction limit. This sub-diffraction localization precision is theoretically bounded by the number of photons emitted per molecule and by the sensor noise. These parameters can be estimated from the raw images. Alternatively, the resolution can be estimated from a rendered image of the localizations. Here, we show how the rendering of localization datasets can influence the resolution estimation based on decorrelation analysis. We demonstrate that a modified histogram rendering, termed bilinear histogram, circumvents the biases introduced by Gaussian or standard histogram rendering. We propose a parameter-free processing pipeline and show that the resolution estimation becomes a function of the localization density and the localization precision, on both simulated and state-of-the-art experimental datasets.

scholarly journals Photon yield enhancement of red fluorophores at cryogenic temperatures

2018 ◽  
Author(s):  
Christiaan N. Hulleman ◽  
Weixing Li ◽  
Ingo Gregor ◽  
Bernd Rieger ◽  
Jörg Enderlein
Keyword(s):  
Liquid Nitrogen ◽  
Single Molecule ◽  
Room Temperature ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Yield Enhancement ◽  
Cryogenic Temperatures ◽  
Photon Yield ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super-resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen temperatures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 · 106to 11 · 106photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.

scholarly journals Sub-Nanometer Precision using Bayesian Grouping of Localizations

2019 ◽  
Author(s):  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Bernd Rieger ◽  
Ralf Jungmann ◽  
Keith A. Lidke
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Time Course ◽  
Bayesian Method ◽  
A Priori ◽  
Super Resolution ◽  
True Number ◽  
Localization Precision ◽  
Better Than ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy super-resolution methods such as DNA-PAINT and (d)STORM can generate multiple observed localizations over the time course of data acquisition from each dye or binding site that are not a priori assigned to those specific dyes or binding sites. We describe a Bayesian method of grouping and combining localizations from multiple blinking/binding events that can improve localization precision to better than one nanometer. The known statistical distribution of the number of binding/blinking events per dye/docking strand along with the precision of each localization event are used to estimate the true number and location of emitters in closely-spaced clusters.

scholarly journals Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Fluorescence Microscope ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Axial Position ◽  
Localization Microscopy ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

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

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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