scholarly journals Visualizing the native cellular organization by coupling cryofixation with expansion microscopy (Cryo-ExM)

2022 ◽  
Author(s):  
Marine H. Laporte ◽  
Nikolai Klena ◽  
Virginie Hamel ◽  
Paul Guichard
Keyword(s):  
Fluorescence Microscopy ◽  
Gold Standard ◽  
Super Resolution ◽  
Cell Ultrastructure ◽  
Chemical Fixation ◽  
Cellular Organization ◽  
Implementation Challenges ◽  
Native Cell ◽  
Super Resolution Microscopy

AbstractCryofixation has proven to be the gold standard for efficient preservation of native cell ultrastructure compared to chemical fixation, but this approach is not widely used in fluorescence microscopy owing to implementation challenges. Here, we develop Cryo-ExM, a method that preserves native cellular organization by coupling cryofixation with expansion microscopy. This method bypasses artifacts associated with chemical fixation and its simplicity will contribute to its widespread use in super-resolution microscopy.

Detection by fluorescence microscopy of N-aminopeptidases in bacteria using an ICT sensor with multiphoton excitation: Usefulness for super-resolution microscopy

2020 ◽  
Vol 321 ◽  
pp. 128487
Author(s):  
Javier Valverde-Pozo ◽  
Jose M. Paredes ◽  
Carmen Salto-Giron ◽  
Pilar Herrero-Foncubierta ◽  
María D. Giron ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Super Resolution ◽  
Multiphoton Excitation ◽  
Super Resolution Microscopy

scholarly journals Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with L p-regularized image reconstruction

Microscopy ◽  
2018 ◽  
Vol 67 (2) ◽  
pp. 89-98
Author(s):  
Tetsuichi Wazawa ◽  
Yoshiyuki Arai ◽  
Yoshinobu Kawahara ◽  
Hiroki Takauchi ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Time Lapse ◽  
Live Cells ◽  
Polarization Angle ◽  
Microscopy Technique ◽  
Present Technique ◽  
Super Resolution Microscopy ◽  
Power Densities

Abstract Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102–106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70–80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.

scholarly journals Meeting experiments at the diffraction barrier: an in-silico widefield fluorescence microscopy

2021 ◽  
Author(s):  
Subhamoy Mahajan ◽  
Tian Tang
Keyword(s):  
Fluorescence Microscopy ◽  
In Silico ◽  
Super Resolution ◽  
Light Sheet ◽  
Atomic Resolution ◽  
Live Cells ◽  
Two Photon ◽  
Color Combinations ◽  
Super Resolution Microscopy ◽  
Diffraction Barrier

AbstractFluorescence microscopy allows the visualization of live cells and their components, but even with advances in super- resolution microscopy, atomic resolution remains unattainable. On the other hand, molecular simulations (MS) can easily access atomic resolution, but comparison with experimental microscopy images has not been possible. In this work, a novel in-silico widefield fluorescence microscopy is proposed, which reduces the resolution of MS to generate images comparable to experiments. This technique will allow cross-validation and compound the knowledge gained from experiments and MS. We demonstrate that in-silico images can be produced with different optical axis, object focal planes, exposure time, color combinations, resolution, brightness and amount of out-of-focus fluorescence. This allows the generation of images that resemble those obtained from widefield, confocal, light-sheet, two-photon and super-resolution microscopy. This technique not only can be used as a standalone visualization tool for MS, but also lays the foundation for other in-silico microscopy methods.

scholarly journals The endoplasmic reticulum adopts two distinct tubule forms

2021 ◽  
Author(s):  
Bowen Wang ◽  
Zhiheng Zhao ◽  
Michael Xiong ◽  
Rui Yan ◽  
Ke Xu
Keyword(s):  
Endoplasmic Reticulum ◽  
Super Resolution ◽  
Cell Types ◽  
Membrane Curvature ◽  
Parallel Lines ◽  
Native Cell ◽  
Functional Implications ◽  
Different Cell Types ◽  
Super Resolution Microscopy ◽  
Er Membrane

The endoplasmic reticulum (ER) is a versatile organelle with diverse functions. Through super-resolution microscopy, we show that the peripheral ER in the mammalian cell adopts two distinct forms of tubules. Whereas an ultrathin form, R1, is consistently covered by ER-membrane curvature-promoting proteins, e.g., Rtn4 in the native cell, in the second form, R2, Rtn4 and analogs are arranged into two parallel lines at a conserved separation of ~105 nm over long ranges. The two tubule forms together account for ~90% of the total tubule length in the cell, with either one being dominant in different cell types. The R1-R2 dichotomy and the final tubule geometry are both co-regulated by Rtn4 (and analogs) and the ER sheet-maintaining protein Climp63, which respectively define the edge curvature and lumen height of the R2 tubules to generate a ribbon-like structure of well-defined width. Accordingly, the R2 tubule width correlates positively with the Climp63 intralumenal size. The R1 and R2 tubules undergo active remodeling at the second/sub-second time scales as they differently accommodate proteins, with the former effectively excluding ER-luminal proteins and ER-membrane proteins with large intraluminal domains. We thus uncover a dynamic structural dichotomy for ER tubules with intriguing functional implications.

scholarly journals Beginner’s guide to producing super-resolved images on a widefield fluorescence microscope

2020 ◽  
Vol 42 (4) ◽  
pp. 52-56
Author(s):  
Ilijana Vojnovic ◽  
Ulrike Endesfelder
Keyword(s):  
Fluorescence Microscopy ◽  
Sample Preparation ◽  
Single Molecule ◽  
Life Sciences ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Specialized Hardware ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

The development of super-resolution microscopy techniques, which are able to achieve resolutions in the nanometre range and as such allow the visualization of subcellular structures and dynamics, has considerably expanded the possibilities of fluorescence microscopy in the life sciences. While a majority of these techniques require highly specialized hardware, single-molecule localization microscopy (SMLM) can be implemented on conventional widefield fluorescence microscopes. Here, we describe what technical upgrades are necessary and discuss some of the difficulties that can be encountered during sample preparation and imaging.

scholarly journals Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

2018 ◽  
Author(s):  
Pereira Pedro M. ◽  
David Albrecht ◽  
Caron Jacobs ◽  
Mark Marsh ◽  
Jason Mercer ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Single Molecule ◽  
Super Resolution ◽  
Receptor Imaging ◽  
Chemical Fixation ◽  
Membrane Organization ◽  
Localization Microscopy ◽  
The World ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

scholarly journals Advanced Methods in Fluorescence Microscopy

2013 ◽  
Vol 36 (1-2) ◽  
pp. 5-17 ◽  
Author(s):  
Luke Fritzky ◽  
David Lagunoff
Keyword(s):  
Fluorescence Microscopy ◽  
Laser Scanning ◽  
Fluorescent Proteins ◽  
Second Harmonic ◽  
Super Resolution ◽  
Good Deal ◽  
Light Sheet ◽  
Confocal Laser ◽  
Will Power ◽  
Super Resolution Microscopy

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

Pushing the super-resolution limit: recent improvements in microscopy below the diffraction limit

2021 ◽  
Author(s):  
D. J. Nieves ◽  
M. A. B. Baker
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Information ◽  
Biological Systems ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Resolving Power ◽  
Resolution Limit ◽  
Single Protein ◽  
Super Resolution Microscopy ◽  
Near Future

Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the ‘super-resolution’ limit and what opportunities remain for further improvements in the near future.

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