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 ◽  
...  
Microscopy ◽  
2018 ◽  
Vol 67 (2) ◽  
pp. 89-98
Author(s):  
Tetsuichi Wazawa ◽  
Yoshiyuki Arai ◽  
Yoshinobu Kawahara ◽  
Hiroki Takauchi ◽  
Takashi Washio ◽  
...  

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.


2021 ◽  
Author(s):  
Subhamoy Mahajan ◽  
Tian Tang

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.


2020 ◽  
Vol 42 (4) ◽  
pp. 52-56
Author(s):  
Ilijana Vojnovic ◽  
Ulrike Endesfelder

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.


2013 ◽  
Vol 36 (1-2) ◽  
pp. 5-17 ◽  
Author(s):  
Luke Fritzky ◽  
David Lagunoff

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.


2022 ◽  
Author(s):  
Marine H. Laporte ◽  
Nikolai Klena ◽  
Virginie Hamel ◽  
Paul Guichard

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.


Author(s):  
D. J. Nieves ◽  
M. A. B. Baker

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.


Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>


Sign in / Sign up

Export Citation Format

Share Document