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 Growth factor dependent changes in nanoscale architecture of focal adhesions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karin Legerstee ◽  
Tsion E. Abraham ◽  
Wiggert A. van Cappellen ◽  
Alex L. Nigg ◽  
Johan A. Slotman ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Intermediate Layer ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Bottom Layer ◽  
Vertical Axis ◽  
Longitudinal Distribution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Mediate Cell

AbstractFocal adhesions (FAs) are flat elongated structures that mediate cell migration and link the cytoskeleton to the extracellular matrix. Along the vertical axis FAs were shown to be composed of three layers. We used structured illumination microscopy to examine the longitudinal distribution of four hallmark FA proteins, which we also used as markers for these layers. At the FA ends pointing towards the adherent membrane edge (heads), bottom layer protein paxillin protruded, while at the opposite ends (tails) intermediate layer protein vinculin and top layer proteins zyxin and VASP extended further. At the tail tips, only intermediate layer protein vinculin protruded. Importantly, head and tail compositions were altered during HGF-induced scattering with paxillin heads being shorter and zyxin tails longer. Additionally, FAs at protruding or retracting membrane edges had longer paxillin heads than FAs at static edges. These data suggest that redistribution of FA-proteins with respect to each other along FAs is involved in cell movement.

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

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.

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

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.

Laser scanning saturated structured illumination microscopy based on phase modulation

2017 ◽  
Vol 396 ◽  
pp. 261-266 ◽  
Author(s):  
Yujia Huang ◽  
Dazhao Zhu ◽  
Luhong Jin ◽  
Cuifang Kuang ◽  
Yingke Xu ◽  
...  
Keyword(s):  
Phase Modulation ◽  
Laser Scanning ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

scholarly journals Laser-free super-resolution microscopy

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Kirti Prakash
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Correlative Imaging ◽  
Deep Blue ◽  
Meiotic Chromosomes ◽  
Set Up ◽  
Super Resolution Microscopy

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope set-up and a mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localization microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as ‘blinking’), detected and localized. The use of a short burst of deep blue excitation (350–380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that stimulated emission depletion and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

scholarly journals Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Christian Pilger ◽  
Jakub Pospíšil ◽  
Marcel Müller ◽  
Martin Ruoff ◽  
Martin Schütte ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Super Resolution ◽  
Image Resolution ◽  
Laser Scanning Microscopy ◽  
Tissue Imaging ◽  
Source Image ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Fluorescence Excitation ◽  
Two Photon

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.

Sign in / Sign up

Export Citation Format

Share Document