Theoretical assessment of two-dimensional nonlinear structured illumination microscopy based on structured excitation and structured stimulated emission depletion

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.

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Optical Microscopy and the Extracellular Matrix Structure: A Review

Cells ◽

10.3390/cells10071760 ◽

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.

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Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

10.21203/rs.3.rs-68002/v1 ◽

2020 ◽

Author(s):

Jiji Chen ◽

Hideki Sasaki ◽

Hoyin Lai ◽

Yijun Su ◽

Jiamin Liu ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Network Performance ◽

Resolution Enhancement ◽

Three Dimensional ◽

Ground Truth ◽

Time Lapse ◽

Stimulated Emission ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Attention Networks

Abstract We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of ~1.4-fold laterally and ~3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

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RNA polymerase II clusters form in line with liquid phase wetting of chromatin

10.1101/2021.02.03.429626 ◽

2021 ◽

Author(s):

Agnieszka Pancholi ◽

Tim Klingberg ◽

Weichun Zhang ◽

Roshan Prizak ◽

Irina Mamontova ◽

...

Keyword(s):

Liquid Phase ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Kinetic Monte Carlo ◽

Monte Carlo Model ◽

Stimulated Emission ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Pol Ii ◽

Phase Wetting

AbstractIt is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state and subsequent pause release to begin transcript elongation. Pol II associates with macromolecular clusters during recruitment, but it remains unclear how Pol II recruitment and pause release might affect these clusters. Here, we show that clusters exhibit morphologies that are in line with wetting of chromatin by a liquid phase enriched in recruited Pol II. Applying instantaneous structured illumination microscopy and stimulated emission double depletion microscopy to pluripotent zebrafish embryos, we find recruited Pol II associated with large clusters, and elongating Pol II with dispersed clusters. A lattice kinetic Monte Carlo model representing recruited Pol II as a liquid phase reproduced the observed cluster morphologies. In this model, chromatin is a copolymer chain containing regions that attract or repel recruited Pol II, supporting droplet formation by wetting or droplet dispersal, respectively.

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