scholarly journals Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/γ-secretase at the cell surface

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Abril Angélica Escamilla-Ayala ◽  
Ragna Sannerud ◽  
Magali Mondin ◽  
Karin Poersch ◽  
Wendy Vermeire ◽  
...  
Keyword(s):  
Cell Surface ◽  
Super Resolution ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Directed Motility ◽  
Fluorescent Tagging ◽  
Super Resolution Microscopy ◽  
High Track

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer’s disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting ‘hotspots’ or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.

scholarly journals CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication

2019 ◽  
Vol 159 (1) ◽  
pp. 48-53 ◽  
Author(s):  
Alžběta Němečková ◽  
Christina Wäsch ◽  
Veit Schubert ◽  
Takayoshi Ishii ◽  
Eva Hřibová ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromatin Structure ◽  
Super Resolution ◽  
Dna Repeats ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Replication Sites ◽  
And Function ◽  
Super Resolution Microscopy

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.

scholarly journals Structured illumination microscopy and its new developments

2016 ◽  
Vol 09 (03) ◽  
pp. 1630010 ◽  
Author(s):  
Jianling Chen ◽  
Caimin Qiu ◽  
Minghai You ◽  
Xiaogang Chen ◽  
Hongqin Yang ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Spatial Resolution ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Living Cells ◽  
The Other ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Recent Developments ◽  
Super Resolution Microscopy

Optical microscopy allows us to observe the biological structures and processes within living cells. However, the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light diffraction. Structured illumination microscopy (SIM), a type of new emerging super-resolution microscopy, doubles the spatial resolution by illuminating the specimen with a patterned light, and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy. In addition, SIM is easier to combine with the other imaging techniques to improve their imaging resolution, leading to the developments of diverse types of SIM. SIM has great potential to meet the various requirements of living cells imaging. Here, we review the recent developments of SIM and its combination with other imaging techniques.

scholarly journals Super-Resolution Tracking of Mitochondrial Dynamics with An Iridium(III) Luminophore

2018 ◽  
Author(s):  
Qixin Chen ◽  
Chengzhi Jin ◽  
Xintian Shao ◽  
Ruilin Guan ◽  
Zhiqi Tian ◽  
...  
Keyword(s):  
Metal Complex ◽  
Transition Metal Complex ◽  
Mitochondrial Dynamics ◽  
Super Resolution ◽  
Living Cells ◽  
Cell Permeability ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Super Resolution Microscopy

AbstractCombining luminescent transition metal complex (LTMC) with super-resolution microscopy is an excellent strategy for the long-term visualization of the dynamics of subcellular structures in living cells. However, it remains unclear whether iridium(III) complexes are applicable for a particular type of super-resolution technique, structured illumination microscopy (SIM), to image subcellular structures.As described herein, we developed an iridium(III) dye, to track mitochondrial dynamics in living cells under SIM. The dye demonstrated excellent specificity and photostability and satisfactory cell permeability. While using SIM to image mitochondria, we achieved an approximately 80-nm resolution that allowed the clear observation of the structure of mitochondrial cristae. We used the dye to monitor and quantify mitochondrial dynamics relative to lysosomes, including fusion involved in mitophagy, and newly discovered mitochondria-lysosome contact (MLC) under different conditions. MLC remained intact and fusion vanished when five receptors, p62, NDP52, OPTN, NBR1, and TAX1BP1, were knocked out, suggesting that these two processes are independence.

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 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.

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 Simultaneous Zn2+ tracking in multiple organelles using super-resolution morphology-correlated organelle identification in living cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hongbao Fang ◽  
Shanshan Geng ◽  
Mingang Hao ◽  
Qixin Chen ◽  
Minglun Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Fluorescent Probe ◽  
Cell Biology ◽  
Pluripotent Stem Cell ◽  
Reliable Method ◽  
Super Resolution ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Turn On

AbstractZn2+ plays important roles in metabolism and signaling regulation. Subcellular Zn2+ compartmentalization is essential for organelle functions and cell biology, but there is currently no method to determine Zn2+ signaling relationships among more than two different organelles with one probe. Here, we report simultaneous Zn2+ tracking in multiple organelles (Zn-STIMO), a method that uses structured illumination microscopy (SIM) and a single Zn2+ fluorescent probe, allowing super-resolution morphology-correlated organelle identification in living cells. To guarantee SIM imaging quality for organelle identification, we develop a new turn-on Zn2+ fluorescent probe, NapBu-BPEA, by regulating the lipophilicity of naphthalimide-derived Zn2+ probes to make it accumulate in multiple organelles except the nucleus. Zn-STIMO with this probe shows that CCCP-induced mitophagy in HeLa cells is associated with labile Zn2+ enhancement. Therefore, direct organelle identification supported by SIM imaging makes Zn-STIMO a reliable method to determine labile Zn2+ dynamics in various organelles with one probe. Finally, SIM imaging of pluripotent stem cell-derived organoids with NapBu-BPEA demonstrates the potential of super-resolution morphology-correlated organelle identification to track biospecies and events in specific organelles within organoids.

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 Dynamic super-resolution structured illumination imaging in the living brain

2019 ◽  
Vol 116 (19) ◽  
pp. 9586-9591 ◽  
Author(s):  
Raphaël Turcotte ◽  
Yajie Liang ◽  
Masashi Tanimoto ◽  
Qinrong Zhang ◽  
Ziwei Li ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Optical Aberrations ◽  
Sample Motion ◽  
Heterogeneous Tissue ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

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