scholarly journals Answers to Fundamental Questions in Superresolution Microscopy

2021 ◽  
Author(s):  
Rainer Heintzmann
Keyword(s):  
Light Microscopy ◽  
Prior Knowledge ◽  
Near Field ◽  
Structured Illumination ◽  
Resolution Limit ◽  
Structured Illumination Microscopy ◽  
Technical Aspects ◽  
Superresolution Microscopy ◽  
Definition Of

This article presents answers to the questions on superresolution and structured illumination microscopy as raised in the editorial of a recent publication [K. Prakash et al. arXiv, 2102.13649, 2021]. The answers are based on my personal views on superresolution in light microscopy, supported by reasoning. Discussed are the definition of superresolution, Abbe’s resolution limit and the classification of superresolution methods into non-linear-, prior-knowledge- and near-field-based superresolution. A further focus is put on capabilities and technical aspects of present and future structured illumination microscopy (SIM) methods.

scholarly journals Answers to fundamental questions in superresolution microscopy

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Rainer Heintzmann
Keyword(s):  
Light Microscopy ◽  
Near Field ◽  
Super Resolution ◽  
Structured Illumination ◽  
Resolution Limit ◽  
Structured Illumination Microscopy ◽  
Technical Aspects ◽  
Superresolution Microscopy ◽  
Definition Of

This article presents answers to the questions on superresolution and structured illumination microscopy (SIM) as raised in the editorial of this collection of articles ( https://doi.org/10.1098/rsta.2020.0143 ). These answers are based on my personal views on superresolution in light microscopy, supported by reasoning. Discussed are the definition of superresolution, Abbe's resolution limit and the classification of superresolution methods into nonlinear-, prior knowledge- and near-field-based superresolution. A further focus is put on the capabilities and technical aspects of present and future SIM methods. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

scholarly journals Double moiré localized plasmon structured illumination microscopy

Nanophotonics ◽  
2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Ruslan Röhrich ◽  
A. Femius Koenderink
Keyword(s):  
Near Field ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Experimental Realization ◽  
Spatial Frequencies ◽  
Wide Range ◽  
Localized Plasmon ◽  
Plasmonic Grating

AbstractStructured illumination microscopy (SIM) is a well-established fluorescence imaging technique, which can increase spatial resolution by up to a factor of two. This article reports on a new way to extend the capabilities of structured illumination microscopy, by combining ideas from the fields of illumination engineering and nanophotonics. In this technique, plasmonic arrays of hexagonal symmetry are illuminated by two obliquely incident beams originating from a single laser. The resulting interference between the light grating and plasmonic grating creates a wide range of spatial frequencies above the microscope passband, while still preserving the spatial frequencies of regular SIM. To systematically investigate this technique and to contrast it with regular SIM and localized plasmon SIM, we implement a rigorous simulation procedure, which simulates the near-field illumination of the plasmonic grating and uses it in the subsequent forward imaging model. The inverse problem, of obtaining a super-resolution (SR) image from multiple low-resolution images, is solved using a numerical reconstruction algorithm while the obtained resolution is quantitatively assessed. The results point at the possibility of resolution enhancements beyond regular SIM, which rapidly vanishes with the height above the grating. In an initial experimental realization, the existence of the expected spatial frequencies is shown and the performance of compatible reconstruction approaches is compared. Finally, we discuss the obstacles of experimental implementations that would need to be overcome for artifact-free SR imaging.

The FDTD Analysis of Near-Field Response for Microgroove Structure With Standing Wave Illumination for the Realization of Coherent Structured Illumination Microscopy

2021 ◽  
Author(s):  
Yizhao Guan ◽  
Hiromasa Kume ◽  
Shotaro Kadoya ◽  
Masaki Michihata ◽  
Satoru Takahashi
Keyword(s):  
Standing Wave ◽  
Incident Angle ◽  
Near Field ◽  
Super Resolution ◽  
Structured Illumination ◽  
Field Phase ◽  
Structured Illumination Microscopy ◽  
Depth Measurement ◽  
Field Response ◽  
Depth Dependency

Abstract Microstructures are widely used in the manufacture of functional surfaces. An optical-based super-resolution, non-invasive method is preferred for the inspection of surfaces with massive microstructures. The Structured Illumination Microscopy (SIM) uses standing-wave illumination to reach optical super-resolution. Recently, coherent SIM is being studied. It can obtain not only the super-resolved intensity distribution but also the phase and amplitude distribution of the sample surface beyond the diffraction limit. By analysis of the phase-depth dependency, the depth measurement for microgroove structures with coherent SIM is expected. FDTD analysis is applied for observing the near-field response of microgroove under the standing-wave illumination. The near-field phase shows depth dependency in this analysis. Moreover, the effects from microgroove width, the incident angle, and the relative position between the standing-wave peak and center of the microgroove are investigated. It is found the near-field phase change can measure depth until 200 nm (aspect ratio 1) with an error of up to 20.4 nm in the case that the microgroove width is smaller than half of the wavelength.

THE FDTD ANALYSIS OF NEAR-FIELD RESPONSE FOR MICROGROOVE STRUCTURE WITH STANDING WAVE ILLUMINATION FOR THE REALIZATION OF COHERENT STRUCTURED ILLUMINATION MICROSCOPY

2021 ◽  
pp. 1-4
Author(s):  
Yizhao Guan ◽  
Hiromasa Kume ◽  
Shotaro Kadoya ◽  
Masaki Michihata ◽  
Satoru Takahashi
Keyword(s):  
Standing Wave ◽  
Incident Angle ◽  
Near Field ◽  
Super Resolution ◽  
Structured Illumination ◽  
Field Phase ◽  
Structured Illumination Microscopy ◽  
Depth Measurement ◽  
Field Response ◽  
Depth Dependency

Abstract Microstructures are widely used in the manufacture of functional surfaces. An optical-based super-resolution, non-invasive method is preferred for the inspection of surfaces with massive microstructures. The Structured Illumination Microscopy (SIM) uses standing-wave illumination to reach optical super-resolution. Recently, coherent SIM is being studied. It can obtain not only the super-resolved intensity distribution but also the phase and amplitude distribution of the sample surface beyond the diffraction limit. By analysis of the phase-depth dependency, the depth measurement for microgroove structures with coherent SIM is expected. FDTD analysis is applied for observing the near-field response of microgroove under the standing-wave illumination. The near-field phase shows depth dependency in this analysis. Moreover, the effects from microgroove width, the incident angle, and the relative position between the standing-wave peak and center of the microgroove are investigated. It is found the near-field phase change can measure depth until 200 nm (aspect ratio 1) with an error of up to 20.4 nm in the case that the microgroove width is smaller than half of the wavelength.

scholarly journals Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

2015 ◽  
Vol 112 (32) ◽  
pp. E4390-E4399 ◽  
Author(s):  
Mathew Stracy ◽  
Christian Lesterlin ◽  
Federico Garza de Leon ◽  
Stephan Uphoff ◽  
Pawel Zawadzki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Spatial Organization ◽  
Search Time ◽  
Population Level ◽  
Bacterial Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Superresolution Microscopy

Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

scholarly journals Structural organization of nuclear lamins A, C, B1, and B2 revealed by superresolution microscopy

2015 ◽  
Vol 26 (22) ◽  
pp. 4075-4086 ◽  
Author(s):  
Takeshi Shimi ◽  
Mark Kittisopikul ◽  
Joseph Tran ◽  
Anne E. Goldman ◽  
Stephen A. Adam ◽  
...  
Keyword(s):  
Structural Organization ◽  
Three Dimensional ◽  
Nuclear Lamina ◽  
Edge Length ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Structural Element ◽  
Superresolution Microscopy ◽  
Nuclear Lamins ◽  
Face Area

The nuclear lamina is a key structural element of the metazoan nucleus. However, the structural organization of the major proteins composing the lamina is poorly defined. Using three-dimensional structured illumination microscopy and computational image analysis, we characterized the supramolecular structures of lamin A, C, B1, and B2 in mouse embryo fibroblast nuclei. Each isoform forms a distinct fiber meshwork, with comparable physical characteristics with respect to mesh edge length, mesh face area and shape, and edge connectivity to form faces. Some differences were found in face areas among isoforms due to variation in the edge lengths and number of edges per face, suggesting that each meshwork has somewhat unique assembly characteristics. In fibroblasts null for the expression of either lamins A/C or lamin B1, the remaining lamin meshworks are altered compared with the lamin meshworks in wild-type nuclei or nuclei lacking lamin B2. Nuclei lacking LA/C exhibit slightly enlarged meshwork faces and some shape changes, whereas LB1-deficient nuclei exhibit primarily a substantial increase in face area. These studies demonstrate that individual lamin isoforms assemble into complex networks within the nuclear lamina and that A- and B-type lamins have distinct roles in maintaining the organization of the nuclear lamina.

scholarly journals Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex

2017 ◽  
Vol 114 (33) ◽  
pp. E6857-E6866 ◽  
Author(s):  
Cori K. Cahoon ◽  
Zulin Yu ◽  
Yongfu Wang ◽  
Fengli Guo ◽  
Jay R. Unruh ◽  
...  
Keyword(s):  
Synaptonemal Complex ◽  
Chromosome Segregation ◽  
Biological Sample ◽  
3D Model ◽  
Three Dimensional ◽  
Optical Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Homologous Chromosomes ◽  
Superresolution Microscopy

The synaptonemal complex (SC), a structure highly conserved from yeast to mammals, assembles between homologous chromosomes and is essential for accurate chromosome segregation at the first meiotic division. In Drosophila melanogaster, many SC components and their general positions within the complex have been dissected through a combination of genetic analyses, superresolution microscopy, and electron microscopy. Although these studies provide a 2D understanding of SC structure in Drosophila, the inability to optically resolve the minute distances between proteins in the complex has precluded its 3D characterization. A recently described technology termed expansion microscopy (ExM) uniformly increases the size of a biological sample, thereby circumventing the limits of optical resolution. By adapting the ExM protocol to render it compatible with structured illumination microscopy, we can examine the 3D organization of several known Drosophila SC components. These data provide evidence that two layers of SC are assembled. We further speculate that each SC layer may connect two nonsister chromatids, and present a 3D model of the Drosophila SC based on these findings.

scholarly journals Concepts for structured illumination microscopy with extended axial resolution through mirrored illumination

2019 ◽  
Author(s):  
James D. Manton ◽  
Florian Ströhl ◽  
Reto Fiolka ◽  
Clemens F. Kaminski ◽  
Eric J. Rees
Keyword(s):  
Confocal Microscopy ◽  
Numerical Aperture ◽  
Wide Field ◽  
Structured Illumination ◽  
Axial Resolution ◽  
Resolution Limit ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
The Cost ◽  
Interferometric Detection

AbstractWide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the resolution limit both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field microscopy. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5M microscopy, have improved the axial resolution beyond that of the lateral, but at the cost of a significantly more complex optical setup. Here, we investigate a simpler dual-objective scheme which we propose can be easily added to an existing 3D-SIM microscope, providing lateral and axial resolutions in excess of 125 nm with conventional fluorophores and without the need for interferometric detection.

scholarly journals Quantitative Analysis of Nuclear Lamins Imaged by Super-Resolution Light Microscopy

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 361 ◽  
Author(s):  
Mark Kittisopikul ◽  
Laura Virtanen ◽  
Pekka Taimen ◽  
Robert D. Goldman
Keyword(s):  
Light Microscopy ◽  
Single Molecule ◽  
Electron Tomography ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Quantitative Information ◽  
Structured Illumination ◽  
Imaging Data ◽  
Structured Illumination Microscopy ◽  
Nuclear Lamins

The nuclear lamina consists of a dense fibrous meshwork of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph-based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information.

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