scholarly journals Low-Power Two-Color Stimulated Emission Depletion Microscopy for Live Cell Imaging

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 330
Author(s):  
Jia Zhang ◽  
Xinwei Gao ◽  
Luwei Wang ◽  
Yong Guo ◽  
Yinru Zhu ◽  
...  
Keyword(s):  
Low Power ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Live Cells ◽  
Sted Microscopy ◽  
Resolution Imaging ◽  
Stimulated Emission Depletion

Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.

scholarly journals The ‘super-resolution’ revolution

2009 ◽  
Vol 37 (5) ◽  
pp. 1042-1044 ◽  
Author(s):  
Ilan Davis
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Light Sheet ◽  
Live Cell ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Light Sheet Microscopy ◽  
Resolution Imaging ◽  
New Instruments

We are currently in the midst of an exciting revolution in microscopy. In many ways, this has been happening for several decades, but it is the rate of development of new methods that has increased recently. The last few years have seen an impressive proliferation of new instruments for imaging at higher resolution, imaging single molecules and faster and more sensitive multidimensional live cell imaging. These include light sheet microscopy, stimulated emission depletion, structured illumination and live cell imaging on the OMX (optical microscopy experimental) platform. However, new probes and image analysis methods have also been crucial for the development of these revolutionary methods.

Live Cell Super-Resolution Imaging with N-SIM

2012 ◽  
Vol 20 (4) ◽  
pp. 18-21
Author(s):  
Christopher B. O'Connell
Keyword(s):  
Light Microscope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Specific Proteins ◽  
Resolution Imaging ◽  
Patterned Illumination

The ability to visualize the distributions of specific proteins with a light microscope and fluorescent probes is largely responsible for our current understanding of cellular structure. A major limitation of this approach arises from the blurring effects of diffraction, which decreases resolution and limits the ability to obtain information at the nanoscale. There has been a tremendous drive to develop optical and computational methods that improve the resolution of the light microscope, and structured illumination microscopy (SIM) is one solution. This method uses patterned illumination to double both lateral and axial resolution. Nikon's N-SIM is a commercial system that integrates the most desirable features of light microscopy, specific labeling of molecules, and live cell imaging, with structured illumination. This provides the ability to achieve super resolution suitable for a range of biological applications.

Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach

Nanoscale ◽  
2018 ◽  
Vol 10 (34) ◽  
pp. 16252-16260 ◽  
Author(s):  
Luwei Wang ◽  
Bingling Chen ◽  
Wei Yan ◽  
Zhigang Yang ◽  
Xiao Peng ◽  
...  
Keyword(s):  
Low Power ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Resolution Limit ◽  
Sted Microscopy ◽  
Microscopy Technique ◽  
Resolution Improvement ◽  
Phasor Plot ◽  
Stimulated Emission Depletion ◽  
Super Resolution Microscopy

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that has achieved significant results in breaking the resolution limit and relevant applications.

scholarly journals Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

2018 ◽  
Vol 217 (4) ◽  
pp. 1537-1552 ◽  
Author(s):  
Sascha Conic ◽  
Dominique Desplancq ◽  
Alexia Ferrand ◽  
Veronique Fischer ◽  
Vincent Heyer ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Cell Types ◽  
Fluorescent Labeling ◽  
Live Cell ◽  
Biological Information ◽  
Live Cells

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.

scholarly journals Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging

2017 ◽  
Vol 67 (2) ◽  
pp. 282-293.e7 ◽  
Author(s):  
Tadasu Nozaki ◽  
Ryosuke Imai ◽  
Mai Tanbo ◽  
Ryosuke Nagashima ◽  
Sachiko Tamura ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Chromatin Domains ◽  
Dynamic Organization

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies

2021 ◽  
Vol 22 (20) ◽  
pp. 11092
Author(s):  
Magalie Bénard ◽  
Damien Schapman ◽  
Christophe Chamot ◽  
Fatéméh Dubois ◽  
Guénaëlle Levallet ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Light Exposure ◽  
Photon Counting ◽  
Live Cell ◽  
Stimulated Emission ◽  
Plot Analysis ◽  
Phasor Plot

Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.

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