scholarly journals High-speed multiplane structured illumination microscopy of living cells using an image-splitting prism

Nanophotonics ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 143-148
Author(s):  
Adrien Descloux ◽  
Marcel Müller ◽  
Vytautas Navikas ◽  
Andreas Markwirth ◽  
Robin van den Eynde ◽  
...  
Keyword(s):  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
Optical Element ◽  
Spatial Light Modulators ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Image Stack ◽  
Light Modulators ◽  
3D Information

AbstractSuper-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live-cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work, we present a multiplane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with high-speed SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally highly resolved 3D image stack. We demonstrate the performance of multiplane SIM by applying this instrument to imaging the dynamics of mitochondria in living COS-7 cells.

scholarly journals High speed multi-plane super-resolution structured illumination microscopy of living cells using an image-splitting prism

2019 ◽  
Author(s):  
Adrien Descloux ◽  
Marcel Müller ◽  
Vytautas Navikas ◽  
Andreas Markwirth ◽  
Robin Van den Eynde ◽  
...  
Keyword(s):  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
Optical Element ◽  
Spatial Light Modulators ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Image Stack ◽  
Light Modulators ◽  
3D Information

Super-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work we present a multi-plane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multi-plane image splitting prism combined with high-speed SR-SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally super-resolved three-dimensional image stack. We demonstrate the performance of multi-plane SR-SIM by applying this instrument to the dynamics of live mitochondrial network.

scholarly journals Super-Resolution Imaging by Dual Iterative Structured Illumination Microscopy

2021 ◽  
Author(s):  
Anna Loeschberger ◽  
Yauheni Novikau ◽  
Ralf Netz ◽  
Marie-Christin Spindler ◽  
Ricardo Benavente ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Brain Slices ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution ◽  
Coated Pits ◽  
Resolution Imaging

Three-dimensional (3D) multicolor super-resolution imaging in the 50-100 nm range in fixed and living cells remains challenging. We extend the resolution of structured illumination microscopy (SIM) by an improved nonlinear iterative reconstruction algorithm that enables 3D multicolor imaging with improved spatiotemporal resolution at low illumination intensities. We demonstrate the performance of dual iterative SIM (diSIM) imaging cellular structures in fixed cells including synaptonemal complexes, clathrin coated pits and the actin cytoskeleton with lateral resolutions of 60-100 nm with standard fluorophores. Furthermore, we visualize dendritic spines in 70 micrometer thick brain slices with an axial resolution < 200 nm. Finally, we image dynamics of the endoplasmatic reticulum and microtubules in living cells with up to 255 frames/s.

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.

scholarly journals GPU-accelerated real-time reconstruction in Python of three-dimensional datasets from structured illumination microscopy with hexagonal patterns

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Hai Gong ◽  
Wenjun Guo ◽  
Mark A. A. Neil
Keyword(s):  
Moving Objects ◽  
Three Dimensional ◽  
Simulated Data ◽  
Super Resolution ◽  
Light Sheet ◽  
Processing Unit ◽  
Structured Illumination ◽  
Reconstruction Algorithms ◽  
Structured Illumination Microscopy ◽  
Axially Moving

We present a structured illumination microscopy system that projects a hexagonal pattern by the interference among three coherent beams, suitable for implementation in a light-sheet geometry. Seven images acquired as the illumination pattern is shifted laterally can be processed to produce a super-resolved image that surpasses the diffraction-limited resolution by a factor of over 2 in an exemplar light-sheet arrangement. Three methods of processing data are discussed depending on whether the raw images are available in groups of seven, individually in a stream or as a larger batch representing a three-dimensional stack. We show that imaging axially moving samples can introduce artefacts, visible as fine structures in the processed images. However, these artefacts are easily removed by a filtering operation carried out as part of the batch processing algorithm for three-dimensional stacks. The reconstruction algorithms implemented in Python include specific optimizations for calculation on a graphics processing unit and we demonstrate its operation on experimental data of static objects and on simulated data of moving objects. We show that the software can process over 239 input raw frames per second at 512 × 512 pixels, generating over 34 super-resolved frames per second at 1024 × 1024 pixels. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

scholarly journals Advances in High-Speed Structured Illumination Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Tianyu Zhao ◽  
Zhaojun Wang ◽  
Tongsheng Chen ◽  
Ming Lei ◽  
Baoli Yao ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Light Power ◽  
Recent Developments ◽  
Super Resolution Microscopy

Super-resolution microscopy surpasses the diffraction limit to enable the observation of the fine details in sub-cellular structures and their dynamics in diverse biological processes within living cells. Structured illumination microscopy (SIM) uses a relatively low illumination light power compared with other super-resolution microscopies and has great potential to meet the demands of live-cell imaging. However, the imaging acquisition and reconstruction speeds limit its further applications. In this article, recent developments all targeted at improving the overall speed of SIM are reviewed. These comprise both hardware and software improvements, which include a reduction in the number of raw images, GPU acceleration, deep learning and the spatial domain reconstruction. We also discuss the application of these developments in live-cell imaging.

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.

scholarly journals Extended mechanical force measurements using structured illumination microscopy

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Kseniya Korobchevskaya ◽  
Huw Colin-York ◽  
Liliana Barbieri ◽  
Marco Fritzsche
Keyword(s):  
Imaging System ◽  
Three Dimensional ◽  
Traction Force ◽  
Super Resolution ◽  
Mechanical Force ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Biological Studies ◽  
Wide Range ◽  
Spatio Temporal

Quantifying cell generated mechanical forces is key to furthering our understanding of mechanobiology. Traction force microscopy (TFM) is one of the most broadly applied force probing technologies, but its sensitivity is strictly dependent on the spatio-temporal resolution of the underlying imaging system. In previous works, it was demonstrated that increased sampling densities of cell derived forces permitted by super-resolution fluorescence imaging enhanced the sensitivity of the TFM method. However, these recent advances to TFM based on super-resolution techniques were limited to slow acquisition speeds and high illumination powers. Here, we present three novel TFM approaches that, in combination with total internal reflection, structured illumination microscopy and astigmatism, improve the spatial and temporal performance in either two-dimensional or three-dimensional mechanical force quantification, while maintaining low illumination powers. These three techniques can be straightforwardly implemented on a single optical set-up offering a powerful platform to provide new insights into the physiological force generation in a wide range of biological studies. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.

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