scholarly journals Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction

2018 ◽  
Author(s):  
Jakub Pospíšil ◽  
Tomáš Lukeš ◽  
Justin Bendesky ◽  
Karel Fliegel ◽  
Kathrin Spendier ◽  
...  
Keyword(s):  
High Resolution ◽  
Open Source ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Live Cells ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
Raw Data ◽  
Bayesian Image Reconstruction

AbstractBackgroundStructured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution).FindingsFive complete and freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods, and with newer Bayesian restoration approaches which we are developing.ConclusionVarious methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments is not typically published. Publicly available, high quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data was processed with SIMToolbox, an open source and freely available software solution for SIM.

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.

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

2019 ◽  
Author(s):  
Karl Johnson ◽  
Guy M. Hagen
Keyword(s):  
Super Resolution ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
High Quality ◽  
Related Data ◽  
Reconstruction Methods ◽  
Hematoxylin And Eosin ◽  
Bayesian Image Reconstruction ◽  
Data Processing Methods

AbstractBackgroundStructured illumination microscopy (SIM) is a method which can be used to image biological samples and can achieve both optical sectioning and super-resolution effects. Optimization of the imaging setup 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.FindingsFive 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 and eosin.ConclusionThe use of fluorescence microscopy is increasing in histopathology. There is a need for methods which reduce artifacts when employing image stitching methods or optical sectioning methods such as SIM. Stitched SIM images produce results which 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.

scholarly journals UCsim2: 2D Structured Illumination Microscopy using UC2

2021 ◽  
Author(s):  
Haoran Wang ◽  
Réne Lachmann ◽  
Barbora Marsikova ◽  
Rainer Heinzmann ◽  
Benedict Diederich
Keyword(s):  
Open Source ◽  
Super Resolution ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
Manufacturing Tolerance ◽  
Resolution Imaging ◽  
Open Source Hardware ◽  
Microscopy Techniques ◽  
Diffraction Barrier

State-of-the-art microscopy techniques enable the imaging of sub-diffraction barrier biological structures at the price of high-costs or lacking transparency. We try to reduce some of these barriers by presenting a super-resolution upgrade to our recently presented open-source optical toolbox UC2. Our new injection moulded parts allow larger builds with higher precision. The 4x lower manufacturing tolerance compared to 3D printing makes assemblies more reproducible. By adding consumer-grade available open-source hardware such as digital mirror devices (DMD) and laser projectors we demonstrate a compact 3D multimodal setup that combines image scanning microscopy (ISM) and structured illumination microscopy (SIM). We demonstrate a gain in resolution and optical sectioning using the two different modes compared to the widefield limit by imaging Alexa Fluor 647- and SiR-stained HeLa cells. We compare different objective lenses and by sharing the designs and manuals of our setup, we make super-resolution imaging available to everyone.

scholarly journals Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Karl Zhanghao ◽  
Xingye Chen ◽  
Wenhui Liu ◽  
Meiqi Li ◽  
Yiqiong Liu ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Imaging Modality ◽  
Super Resolution ◽  
Vital Role ◽  
Molecular Structures ◽  
Polarization Microscopy ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging

Abstract Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

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.

scholarly journals Fringe optimization for structured illumination super-resolution microscope with digital micromirror device

2019 ◽  
Vol 12 (03) ◽  
pp. 1950014 ◽  
Author(s):  
Xibin Yang ◽  
Qian Zhu ◽  
Zhenglong Sun ◽  
Gang Wen ◽  
Xin Jin ◽  
...  
Keyword(s):  
Modulation Depth ◽  
Low Cost ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Digital Micromirror Device ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Wide Range ◽  
Fringe Patterns

Structured illumination microscopy (SIM) is a promising super-resolution technique for imaging subcellular structures and dynamics due to its compatibility with most commonly used fluorescent labeling methods. Structured illumination can be obtained by either laser interference or projection of fringe patterns. Here, we proposed a fringe projector composed of a compact multi-wavelength LEDs module and a digital micromirror device (DMD) which can be directly attached to most commercial inverted fluorescent microscopes and update it into a SIM system. The effects of the period and duty cycle of fringe patterns on the modulation depth of the structured light field were studied. With the optimized fringe pattern, [Formula: see text] resolution improvement could be obtained with high-end oil objectives. Multicolor imaging and dynamics of subcellular organelles in live cells were also demonstrated. Our method provides a low-cost solution for SIM setup to expand its wide range of applications to most research labs in the field of life science and medicine.

scholarly journals Extending resolution of structured illumination microscopy with sparse deconvolution

2021 ◽  
Author(s):  
Weisong Zhao ◽  
Shiqun Zhao ◽  
Liuju Li ◽  
Xiaoshuai Huang ◽  
Shijia Xing ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cell ◽  
Frame Rate ◽  
Live Cells ◽  
Photon Flux ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution

Abstract The spatial resolutions of live-cell super-resolution microscopes are limited by the maximum collected photon flux. Taking advantage of a priori knowledge of the sparsity and continuity of biological structures, we develop a deconvolution algorithm that further extends the resolution of super-resolution microscopes under the same photon budgets by nearly twofold. As a result, sparse structured illumination microscopy (Sparse-SIM) achieves ~60 nm resolution at a 564 Hz frame rate, allowing it to resolve intricate structural intermediates, including small vesicular fusion pores, ring-shaped nuclear pores formed by different nucleoporins, and relative movements between the inner and outer membranes of mitochondria in live cells. Likewise, sparse deconvolution can be used to increase the three-dimensional resolution and contrast of spinning-disc confocal-based SIM (SD-SIM), and operates under conditions with the insufficient signal-to-noise-ratio, all of which allows routine four-color, three-dimensional, ~90 nm resolution live-cell super-resolution imaging. Overall, sparse deconvolution may be a general tool to push the spatiotemporal resolution limits of live-cell fluorescence microscopy.

A Platinum(II) Based Correlative Probe for Bacteria

2019 ◽  
Author(s):  
Anna Maria Ranieri ◽  
Kathryn Leslie ◽  
Song Huang ◽  
Stefano Stagni ◽  
Denis Jacquemin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Super Resolution ◽  
Molecular Probes ◽  
Live Cells ◽  
Structured Illumination ◽  
Luminescent Probe ◽  
Structured Illumination Microscopy ◽  
Cellular Localisation ◽  
Super Resolution Microscopy ◽  
Nanosims Analysis

There is a lack of molecular probes for imaging bacteria, in comparison to the array of such tools available for the imaging of mammalian cells. This is especially so for correlative probes, which are proving to be powerful tools for enhancing the imaging of live cells. In this work a platinum(II)-naphthalimide molecule has been developed to extend small molecule correlative probes to bacterial imaging. The probe was designed to exploit the naphthalimide moiety as a luminescent probe for super-resolution microscopy, with the platinum(II) centre enabling visualisation of the complex with ion nanoscopy. Photophysical characterisation and theoretical studies confirmed that the emission properties of the naphthalimide are not altered by the platinum(II) centre. Structured illumination microscopy (SIM) imaging on live <i>Bacillus cereus</i>revealed that the platinum(II) centre does not change the sub-cellular localisation of the naphthalimide, and confirmed the suitability of the probe for super-resolution microscopy. NanoSIMS analysis of the sample was used to monitor the uptake of the platinum(II) complex within the bacteria and proved the correlative action of the probe. The successful combination of these two probe moieties with no perturbation of their individual detection introduces a platform for a versatile range of new correlative probes for bacteria.

scholarly journals mmSIM: an open toolbox for accessible structured illumination microscopy

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Craig T. Russell ◽  
Michael Shaw
Keyword(s):  
Open Source ◽  
Open Source Software ◽  
High Speed ◽  
Super Resolution ◽  
Software Tools ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Light Modulator ◽  
Wide Range ◽  
Hardware Designs

Since the first practical super-resolution structured illumination fluorescence microscopes (SIM) were demonstrated more than two decades ago, the method has become increasingly popular for a wide range of bioimaging applications. The high cost and relative inflexibility of commercial systems, coupled with the conceptual simplicity of the approach and the desire to exploit and customize existing hardware, have led to the development of a large number of home-built systems. Several detailed hardware designs are available in the scientific literature, complemented by open-source software tools for SIM image validation and reconstruction. However, there remains a lack of simple open-source software to control these systems and manage the synchronization between hardware components, which is critical for effective SIM imaging. This article describes a new suite of software tools based on the popular Micro-Manager package, which enable the keen microscopist to develop and run a SIM system. We use the software to control two custom-built, high-speed, spatial light modulator-based SIM systems, evaluating their performance by imaging a range of fluorescent samples. By simplifying the process of SIM hardware development, we aim to support wider adoption of the technique. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

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