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.

Generation and Control of Wide-Field Three-Dimensional Structured Illumination for Advanced Microscopic Imaging

2012 ◽  
Vol 516 ◽  
pp. 640-644
Author(s):  
Shin Usuki ◽  
Hiroyoshi Kanaka ◽  
Kenjiro Takai Miura
Keyword(s):  
Three Dimensional ◽  
Fluorescent Imaging ◽  
Wide Field ◽  
Structured Illumination ◽  
Axial Resolution ◽  
Structured Illumination Microscopy ◽  
Spatial Control ◽  
Microscopic Imaging ◽  
Point Spread ◽  
Resolution Improvement

In a variety of practical microscopic imaging applications, many industries require not only lateral resolution improvement but also axial resolution improvement. The resolution in optical microscopy is limited by diffraction and determined by the wavelength of the incident light and the numerical aperture (NA) of the objective lens. The diffraction limit is mathematically described by a point spread function in the imaging system, and three-dimensional (3D) point spread functions describe both the lateral and axial resolutions. Thus, it is useful to focus on exceeding this limit and improving the resolution of optical imaging by the spatial control of structured illumination. Structured illumination microscopy is a familiar technique to improve resolution in fluorescent imaging, and it is expected to be applied to industrial applications. Microscopic imaging is convenient, non-destructive, and has a high-throughput performance and compatibility with a number of applications. However, the spatial resolution of conventional light microscopy is limited to wavelength scale and the depth of field is shallow; hence, it is difficult to obtain detailed 3D spatial data of the object to be measured. Here, we propose a new technique for generating and controlling wide-field 3D structured illumination. The technique, based on the 3D interference of multiple laser beams, provides lateral and axial resolution improvement, and a wide 3D field of view. The spatial configuration of the beams was theoretically examined and the optimal incident angle of the multiple beams was confirmed. Numerical simulations using the finite difference time domain (FDTD) method were carried out and confirmed the generation of 3D structured illumination and spatial control of the illumination by using the phase shift of incident beams.

scholarly journals Comparison of Two- and Three-Beam Interference Pattern Generation in Structured Illumination Microscopy

Photonics ◽  
2021 ◽  
Vol 8 (12) ◽  
pp. 526
Author(s):  
Jiuling Liao ◽  
Lina Liu ◽  
Tingai Chen ◽  
Xianyuan Xia ◽  
Hui Li ◽  
...  
Keyword(s):  
Fringe Pattern ◽  
Three Dimensional ◽  
Pattern Generation ◽  
Wide Field ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
Imaging Results ◽  
Dynamic Target ◽  
Beam Patterns

Structured illumination microscopy (SIM) provides wide-field optical sectioning in the focal plane by modulating the imaging information using fringe pattern illumination. For generating the fringe pattern illumination, a digital micro-mirror device (DMD) is commonly used due to its flexibility and fast refresh rate. However, the benefit of different pattern generation, for example, the two-beam interference mode and the three-beam interference mode, has not been clearly investigated. In this study, we systematically analyze the optical sectioning provided by the two-beam inference mode and the three-beam interference mode of DMD. The theoretical analysis and imaging results show that the two-beam interference mode is suitable for fast imaging of the superficial dynamic target due to reduced number of phase shifts needed to form the image, and the three-beam interference mode is ideal for imaging three-dimensional volume due to its superior optical sectioning by the improved modulation of the illumination patterns. These results, we believe, will provide better guidance for the use of DMD for SIM imaging and also for the choice of beam patterns in SIM application in the future.

scholarly journals Optical Microscopy and the Extracellular Matrix Structure: A Review

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1760
Author(s):  
Joshua J. A. Poole ◽  
Leila B. Mostaço-Guidolin
Keyword(s):  
Extracellular Matrix ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Biological Tissues ◽  
The Body ◽  
Stimulated Emission ◽  
Substantial Part ◽  
Fluorescence Lifetime Imaging ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

Biological tissues are not uniquely composed of cells. A substantial part of their volume is extracellular space, which is primarily filled by an intricate network of macromolecules constituting the extracellular matrix (ECM). The ECM serves as the scaffolding for tissues and organs throughout the body, playing an essential role in their structural and functional integrity. Understanding the intimate interaction between the cells and their structural microenvironment is central to our understanding of the factors driving the formation of normal versus remodelled tissue, including the processes involved in chronic fibrotic diseases. The visualization of the ECM is a key factor to track such changes successfully. This review is focused on presenting several optical imaging microscopy modalities used to characterize different ECM components. In this review, we describe and provide examples of applications of a vast gamut of microscopy techniques, such as widefield fluorescence, total internal reflection fluorescence, laser scanning confocal microscopy, multipoint/slit confocal microscopy, two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG, THG), coherent anti-Stokes Raman scattering (CARS), fluorescence lifetime imaging microscopy (FLIM), structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), ground-state depletion microscopy (GSD), and photoactivated localization microscopy (PALM/fPALM), as well as their main advantages, limitations.

scholarly journals Linear elements are stable structures along the chromosome axis in fission yeast meiosis

Chromosoma ◽  
2021 ◽  
Author(s):  
Da-Qiao Ding ◽  
Atsushi Matsuda ◽  
Kasumi Okamasa ◽  
Yasushi Hiraoka
Keyword(s):  
Fission Yeast ◽  
Strand Break ◽  
Wide Field ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Meiotic Chromosomes ◽  
Linear Elements ◽  
Yeast Meiosis ◽  
Chromosome Axis ◽  
Stable Structures

AbstractThe structure of chromosomes dramatically changes upon entering meiosis to ensure the successful progression of meiosis-specific events. During this process, a multilayer proteinaceous structure called a synaptonemal complex (SC) is formed in many eukaryotes. However, in the fission yeast Schizosaccharomyces pombe, linear elements (LinEs), which are structures related to axial elements of the SC, form on the meiotic cohesin-based chromosome axis. The structure of LinEs has been observed using silver-stained electron micrographs or in immunofluorescence-stained spread nuclei. However, the fine structure of LinEs and their dynamics in intact living cells remain to be elucidated. In this study, we performed live cell imaging with wide-field fluorescence microscopy as well as 3D structured illumination microscopy (3D-SIM) of the core components of LinEs (Rec10, Rec25, Rec27, Mug20) and a linE-binding protein Hop1. We found that LinEs form along the chromosome axis and elongate during meiotic prophase. 3D-SIM microscopy revealed that Rec10 localized to meiotic chromosomes in the absence of other LinE proteins, but shaped into LinEs only in the presence of all three other components, the Rec25, Rec27, and Mug20. Elongation of LinEs was impaired in double-strand break-defective rec12− cells. The structure of LinEs persisted after treatment with 1,6-hexanediol and showed slow fluorescence recovery from photobleaching. These results indicate that LinEs are stable structures resembling axial elements of the SC.

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 A technique for resolution assessment in blind-SIM experiments

2021 ◽  
Author(s):  
Imen Boujmil ◽  
Giancarlo Ruocco ◽  
Marco Leonetti
Keyword(s):  
High Resolution ◽  
Biological Sample ◽  
A Priori ◽  
Resolution Enhancement ◽  
Super Resolution ◽  
Experimental Setup ◽  
Wide Field ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Measurement

Super resolution techniques are an excellent alternative to wide field microscopy, providing high resolution also in (typically fragile) biological sample. Among the various super resolution techniques, Structured Illumination Microscopy (SIM) improve resolution by employing multiple illumination patterns to be deconvolved with a dedicated software. In the case of blind SIM techniques, unknown patterns, such as speckles, are used, thus providing super resolved images, nearly unaffected by aberrations with a simplified experimental setup. Scattering Assisted Imaging, a special blind SIM technique, exploits an illumination PSF (speckle grains size), smaller than the collection PSF (defined by the collection objectives), to surpass the typical SIM resolution enhancement. However, if SAI is used, it is very difficult to extract the resolution enhancement form a priori considerations. In this paper we propose a protocol and experimental setup for the resolution measurement, demonstrating the resolution enhancement for different collection PSF values.

Multiphoton 3D structured illumination microscopy for enhanced axial resolution in deep imaging

2015 ◽  
Author(s):  
Qiyuan Song ◽  
Keisuke Isobe ◽  
Fumihiko Kannari ◽  
Hiroyuki Kawano ◽  
Akiko Kumagai ◽  
...  
Keyword(s):  
Structured Illumination ◽  
Axial Resolution ◽  
Structured Illumination Microscopy ◽  
Deep Imaging

scholarly journals Artifact-free whole-slide imaging with structured illumination microscopy and Bayesian image reconstruction

GigaScience ◽  
2020 ◽  
Vol 9 (4) ◽  
Author(s):  
Karl A Johnson ◽  
Guy M Hagen
Keyword(s):  
Super Resolution ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
High Quality ◽  
Related Data ◽  
Reconstruction Methods ◽  
Set Up ◽  
Bayesian Image Reconstruction ◽  
Hematoxylin Eosin

Abstract Background Structured illumination microscopy (SIM) is a method that can be used to image biological samples and can achieve both optical sectioning and super-resolution effects. Optimization of the imaging set-up and data-processing methods results in high-quality images without artifacts due to mosaicking or due to the use of SIM methods. Reconstruction methods based on Bayesian estimation can be used to produce images with a resolution beyond that dictated by the optical system. Findings Five complete datasets are presented including large panoramic SIM images of human tissues in pathophysiological conditions. Cancers of the prostate, skin, ovary, and breast, as well as tuberculosis of the lung, were imaged using SIM. The samples are available commercially and are standard histological preparations stained with hematoxylin-eosin. Conclusion The use of fluorescence microscopy is increasing in histopathology. There is a need for methods that reduce artifacts caused by the use of image-stitching methods or optical sectioning methods such as SIM. Stitched SIM images produce results that may be useful for intraoperative histology. Releasing high-quality, full-slide images and related data will aid researchers in furthering the field of fluorescent histopathology.

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