scholarly journals 3D super-resolution imaging using a generalized and scalable progressive refinement method on sparse recovery (PRIS)

2019 ◽  
Author(s):  
Xiyu Yi ◽  
Rafael Piestun ◽  
Shimon Weiss
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Super Resolution ◽  
Sparse Recovery ◽  
Two Dimensions ◽  
High Density ◽  
Three Dimensions ◽  
Spatial Density ◽  
Progressive Refinement ◽  
Refinement Method

ABSTRACTWithin the family of super-resolution (SR) fluorescence microscopy, single-molecule localization microscopies (PALM[1], STORM[2] and their derivatives) afford among the highest spatial resolution (approximately 5 to 10 nm), but often with moderate temporal resolution. The high spatial resolution relies on the adequate accumulation of precise localizations of bright fluorophores, which requires the bright fluorophores to possess a relatively low spatial density. Several methods have demonstrated localization at higher densities in both two dimensions (2D)[3, 4] and three dimensions (3D)[5-7]. Additionally, with further advancements, such as functional super-resolution[8, 9] and point spread function (PSF) engineering with[8-11] or without[12] multi-channel observations, extra information (spectra, dipole orientation) can be encoded and recovered at the single molecule level. However, such advancements are not fully extended for high-density localizations in 3D. In this work, we adopt sparse recovery using simple matrix/vector operations, and propose a systematic progressive refinement method (dubbed as PRIS) for 3D high-density reconstruction. Our method allows for localization reconstruction using experimental PSFs that include the spatial aberrations and fingerprint patterns of the PSFs[13]. We generalized the method for PSF engineering, multi-channel and multi-species observations using different forms of matrix concatenations. Reconstructions with both double-helix and astigmatic PSFs, for both single and biplane settings are demonstrated, together with the recovery capability for a mixture of two different color species.

scholarly journals STORM Offers Super-Resolution in 3D!

2008 ◽  
Vol 16 (6) ◽  
pp. 3-5
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Spatial Resolution ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Substantial Improvement ◽  
Two Dimensions ◽  
Three Dimensions ◽  
Optical Methods ◽  
Axial Dimension ◽  
Diffraction Barrier ◽  
Better Than

For the first few centuries of microscopy, spatial resolution was limited by the diffraction barrier. Recently, this barrier has been broken using several different methods. Optical methods that provide better resolution than the diffraction barrier are referred to as super-resolution. Although these techniques have significantly improved resolution in two dimensions (x and y) or in the axial dimension (z), it has not been possible to achieve substantial improvement in all three dimensions simultaneously. A study by Bo Huang, Wenqin Wang, Mark Bates, and Xiaowei Zhuang demonstrated a breakthrough by achieving a spatial resolution that is 10 times better than the diffraction limit in all three dimensions without using sample or optical-beam scanning.

scholarly journals 3D clustering analysis of super-resolution microscopy data by 3D Voronoi tessellations

2017 ◽  
Author(s):  
Leonid Andronov ◽  
Jonathan Michalon ◽  
Khalid Ouararhni ◽  
Igor Orlov ◽  
Ali Hamiche ◽  
...  
Keyword(s):  
Single Molecule ◽  
Local Density ◽  
3D Structure ◽  
Super Resolution ◽  
Three Dimensions ◽  
3D Analysis ◽  
Voronoi Tessellations ◽  
X Ray Crystallography ◽  
Cellular Context ◽  
Microscopy Data

AbstractSingle-molecule localization microscopy (SMLM) can play an important role in integrated structural biology approaches for example at the interface of cryo electron microscopy (cryo-EM), X-ray crystallography, NMR and fluorescence imaging to identify, localize and determine the 3D structure of cellular structures. While many tools exist for the 3D analysis and visualisation of crystal or cryo-EM structures little exists for 3D SMLM data which can provide fascinating insights but are particularly challenging to analyze in three dimensions especially in a dense cellular context. We developed 3DClusterViSu, a method based on 3D Voronoi tessellations that allows local density estimation, segmentation & quantification of 3D SMLM data and visualization of protein clusters within a 3D tool. We show its robust performance on microtubules and histone proteins H2B and CENP-A with distinct spatial distributions. 3DClusterViSu will favor multi-scale and multi-resolution synergies to allow integrating molecular and cellular levels in the analysis of macromolecular complexes.

scholarly journals Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single Molecule Localization Microscopy

2021 ◽  
Author(s):  
Siddharth Matikonda ◽  
Dominic Helmerich ◽  
Mara Meub ◽  
Gerti Beliu ◽  
Philip Kollmannsberger ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Computational Analysis ◽  
Super Resolution ◽  
Underlying Mechanism ◽  
New Approach ◽  
Localization Microscopy ◽  
Order Of Magnitude ◽  
Resolution Single ◽  
Single Molecule Localization Microscopy

<p>The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multi-step sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.</p>

scholarly journals Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses

2021 ◽  
Vol 13 ◽  
Author(s):  
Gabriella Gagliano ◽  
Tyler Nelson ◽  
Nahima Saliba ◽  
Sofía Vargas-Hernández ◽  
Anna-Karin Gustavsson
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Single Molecule Tracking ◽  
Resolution Imaging

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.

scholarly journals Isotropic Three-Dimensional Dual-Color Super-Resolution Microscopy with Metal-Induced Energy Transfer

2021 ◽  
Author(s):  
Jan Christoph Thiele ◽  
Marvin Jungblut ◽  
Dominic A. Helmerich ◽  
Roman Tsukanov ◽  
Anna Chizhik ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Three Dimensions ◽  
Lateral Resolution ◽  
Cellular Structures ◽  
Axial Resolution ◽  
Dual Color ◽  
Super Resolution Microscopy

Over the last two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution. Similar to conventional optical microscopy, the axial resolution is by a factor three to five worse than the lateral resolution. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging which achieves an axial resolution down to nanometers. It exploits the distance dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of sub-cellular structures. Moreover, we employed spectral demixing for implementing dual-color MIET-dSTORM that allows us to image and co-localize, in three dimensions, two different cellular structures simultaneously.

scholarly journals Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single Molecule Localization Microscopy

2021 ◽  
Author(s):  
Siddharth Matikonda ◽  
Dominic Helmerich ◽  
Mara Meub ◽  
Gerti Beliu ◽  
Philip Kollmannsberger ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Computational Analysis ◽  
Super Resolution ◽  
Underlying Mechanism ◽  
New Approach ◽  
Localization Microscopy ◽  
Order Of Magnitude ◽  
Resolution Single ◽  
Single Molecule Localization Microscopy

<p>The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multi-step sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.</p>

scholarly journals Staircase array of inclined refractive multi-lenses for large field of view pixel super-resolution scanning transmission hard X-ray microscopy

2021 ◽  
Vol 28 (3) ◽  
Author(s):  
Talgat Mamyrbayev ◽  
Katsumasa Ikematsu ◽  
Hidekazu Takano ◽  
Yanlin Wu ◽  
Kenji Kimura ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Super Resolution ◽  
High Energy ◽  
Two Dimensions ◽  
Field Of View ◽  
Large Field ◽  
Acquisition Time ◽  
X Rays ◽  
X Ray ◽  
Scanning Transmission

Owing to the development of X-ray focusing optics during the past decades, synchrotron-based X-ray microscopy techniques allow the study of specimens with unprecedented spatial resolution, down to 10 nm, using soft and medium X-ray photon energies, though at the expense of the field of view (FOV). One of the approaches to increase the FOV to square millimetres is raster-scanning of the specimen using a single nanoprobe; however, this results in a long data acquisition time. This work employs an array of inclined biconcave parabolic refractive multi-lenses (RMLs), fabricated by deep X-ray lithography and electroplating to generate a large number of long X-ray foci. Since the FOV is limited by the pattern height if a single RML is used by impinging X-rays parallel to the substrate, many RMLs at regular intervals in the orthogonal direction were fabricated by tilted exposure. By inclining the substrate correspondingly to the tilted exposure, 378000 X-ray line foci were generated with a length in the centimetre range and constant intervals in the sub-micrometre range. The capability of this new X-ray focusing device was first confirmed using ray-tracing simulations and then using synchrotron radiation at BL20B2 of SPring-8, Japan. Taking account of the fact that the refractive lens is effective for focusing high-energy X-rays, the experiment was performed with 35 keV X-rays. Next, by scanning a specimen through the line foci, this device was used to perform large FOV pixel super-resolution scanning transmission hard X-ray microscopy (PSR-STHXM) with a 780 ± 40 nm spatial resolution within an FOV of 1.64 cm × 1.64 cm (limited by the detector area) and a total scanning time of 4 min. Biomedical implant abutments fabricated via selective laser melting using Ti–6Al–4V medical alloy were measured by PSR-STHXM, suggesting its unique potential for studying extended and thick specimens. Although the super-resolution function was realized in one dimension in this study, it can be expanded to two dimensions by aligning a pair of presented devices orthogonally.

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