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 Two-dimensional TIRF-SIM–traction force microscopy (2D TIRF-SIM-TFM)

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liliana Barbieri ◽  
Huw Colin-York ◽  
Kseniya Korobchevskaya ◽  
Di Li ◽  
Deanna L. Wolfson ◽  
...  
Keyword(s):  
Resolution Enhancement ◽  
Traction Force ◽  
Super Resolution ◽  
Traction Force Microscopy ◽  
Two Dimensional ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Force Microscopy ◽  
Health And Disease ◽  
Spatio Temporal

AbstractQuantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

An overview of structured illumination microscopy: recent advances and perspectives

2021 ◽  
Author(s):  
Krishnendu Samanta ◽  
Joby Joseph
Keyword(s):  
Spatial Frequency ◽  
Basic Principle ◽  
Imaging Techniques ◽  
Resolution Enhancement ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Structured Illumination ◽  
Post Processing ◽  
Structured Illumination Microscopy ◽  
Near Future

Abstract Structured illumination microscopy (SIM) is one of the most significant widefield super-resolution optical imaging techniques. The conventional SIM utilizes a sinusoidal structured pattern to excite the fluorescent sample; which eventually down-modulates higher spatial frequency sample information within the diffraction-limited passband of the microscopy system and provides around two-fold resolution enhancement over diffraction limit after suitable computational post-processing. Here we provide an overview of the basic principle, image reconstruction, technical development of the SIM technique. Nonetheless, in order to push the SIM resolution further towards the extreme nanoscale dimensions, several different approaches are launched apart from the conventional SIM. Among the various SIM methods, some of the important techniques e.g. TIRF, non-linear, plasmonic, speckle SIM etc. are discussed elaborately. Moreover, we highlight different implementations of SIM in various other imaging modalities to enhance their imaging performances with augmented capabilities. Finally, some future outlooks are mentioned which might develop fruitfully and pave the way for new discoveries in near future.

scholarly journals Visualizing ultrastructural details of placental tissue with super-resolution structured illumination microscopy

2020 ◽  
Author(s):  
Luis E. Villegas-Hernández ◽  
Mona Nystad ◽  
Florian Ströhl ◽  
Purusotam Basnet ◽  
Ganesh Acharya ◽  
...  
Keyword(s):  
Cell Biology ◽  
Resolution Enhancement ◽  
Super Resolution ◽  
Placental Tissue ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Chorionic Villi ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded

AbstractSuper-resolution fluorescence microscopy is a widely employed technique in cell biology research, yet remains relatively unexplored in the field of histo-pathology. Here, we describe the sample preparation steps and acquisition parameters necessary to obtain fluorescent multicolor super-resolution structured illumination microscopy (SIM) images of both formalin-fixed paraffin-embedded and cryo-preserved placental tissue sections. We compare super-resolved images of chorionic villi against diffraction-limited deconvolution microscopy and demonstrate the significant contrast and resolution enhancement attainable with SIM. We show that SIM resolves ultrastructural details such as the syncytiotrophoblast’s microvilli brush border, which up until now has been only resolvable by electron microscopy.

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 A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies

2020 ◽  
Vol 12 (535) ◽  
pp. eaay0071 ◽  
Author(s):  
Zhen Liu ◽  
Quynh P. H. Nguyen ◽  
Qingxu Guan ◽  
Alexandra Albulescu ◽  
Lauren Erdman ◽  
...  
Keyword(s):  
Lung Function ◽  
A Priori ◽  
Three Dimensional ◽  
Super Resolution ◽  
Structural Defects ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Motile Cilia ◽  
Optical Reconstruction

Airway clearance of pathogens and particulates relies on motile cilia. Impaired cilia motility can lead to reduction in lung function, lung transplant, or death in some cases. More than 50 proteins regulating cilia motility are linked to primary ciliary dyskinesia (PCD), a heterogeneous, mainly recessive genetic lung disease. Accurate PCD molecular diagnosis is essential for identifying therapeutic targets and for initiating therapies that can stabilize lung function, thereby reducing socioeconomic impact of the disease. To date, PCD diagnosis has mainly relied on nonquantitative methods that have limited sensitivity or require a priori knowledge of the genes involved. Here, we developed a quantitative super-resolution microscopy workflow: (i) to increase sensitivity and throughput, (ii) to detect structural defects in PCD patients’ cells, and (iii) to quantify motility defects caused by yet to be found PCD genes. Toward these goals, we built a localization map of PCD proteins by three-dimensional structured illumination microscopy and implemented quantitative image analysis and machine learning to detect protein mislocalization, we analyzed axonemal structure by stochastic optical reconstruction microscopy, and we developed a high-throughput method for detecting motile cilia uncoordination by rotational polarity. Together, our data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.

Localized plasmon assisted structured illumination microscopy for wide-field high-speed dispersion-independent super resolution imaging

Nanoscale ◽  
2014 ◽  
Vol 6 (11) ◽  
pp. 5807-5812 ◽  
Author(s):  
Joseph Louis Ponsetto ◽  
Feifei Wei ◽  
Zhaowei Liu
Keyword(s):  
Antenna Array ◽  
High Speed ◽  
Super Resolution ◽  
Fluorescent Imaging ◽  
Wide Field ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging ◽  
Localized Plasmon ◽  
Imaging Resolution

Fluorescent imaging resolution down to 51 nm is shown by generating tunable localized plasmon excitations on a nano-antenna array.

Wide Field Super-Resolution Surface Imaging through Plasmonic Structured Illumination Microscopy

Nano Letters ◽  
2014 ◽  
Vol 14 (8) ◽  
pp. 4634-4639 ◽  
Author(s):  
Feifei Wei ◽  
Dylan Lu ◽  
Hao Shen ◽  
Weiwei Wan ◽  
Joseph Louis Ponsetto ◽  
...  
Keyword(s):  
Super Resolution ◽  
Wide Field ◽  
Surface Imaging ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

scholarly journals Video-rate multi-color structured illumination microscopy with simultaneous real-time reconstruction

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Andreas Markwirth ◽  
Mario Lachetta ◽  
Viola Mönkemöller ◽  
Rainer Heintzmann ◽  
Wolfgang Hübner ◽  
...  
Keyword(s):  
Image Reconstruction ◽  
High Speed ◽  
Resolution Enhancement ◽  
Optical Diffraction ◽  
Video Frame ◽  
Wide Field ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Reconstruction Software ◽  
Microscopy Techniques

Abstract Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250 ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics.

scholarly journals Metamaterial assisted illumination nanoscopy via random super-resolution speckles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yeon Ui Lee ◽  
Junxiang Zhao ◽  
Qian Ma ◽  
Larousse Khosravi Khorashad ◽  
Clara Posner ◽  
...  
Keyword(s):  
Surface Characterization ◽  
Low Cost ◽  
Near Field ◽  
Super Resolution ◽  
Diffraction Limit ◽  
High Temporal Resolution ◽  
Wide Field ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Bio Imaging

AbstractStructured illumination microscopy (SIM) is one of the most powerful and versatile optical super-resolution techniques. Compared with other super-resolution methods, SIM has shown its unique advantages in wide-field imaging with high temporal resolution and low photon damage. However, traditional SIM only has about 2 times spatial resolution improvement compared to the diffraction limit. In this work, we propose and experimentally demonstrate an easily-implemented, low-cost method to extend the resolution of SIM, named speckle metamaterial-assisted illumination nanoscopy (speckle-MAIN). A metamaterial structure is introduced to generate speckle-like sub-diffraction-limit illumination patterns in the near field with improved spatial frequency. Such patterns, similar to traditional SIM, are then used to excite objects on top of the surface. We demonstrate that speckle-MAIN can bring the resolution down to 40 nm and beyond. Speckle-MAIN represents a new route for super-resolution, which may lead to important applications in bio-imaging and surface characterization.

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