scholarly journals Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures

2021 ◽  
Vol 22 (19) ◽  
pp. 10194
Author(s):  
Merel Stiekema ◽  
Frans C. S. Ramaekers ◽  
Dimitrios Kapsokalyvas ◽  
Marc A. M. J. van Zandvoort ◽  
Rogier J. A. Veltrop ◽  
...  
Keyword(s):  
Layer Thickness ◽  
Super Resolution ◽  
Dermal Fibroblasts ◽  
Stimulated Emission ◽  
Immunofluorescence Staining ◽  
Fluorescence Labeling ◽  
Confocal Scanning Laser Microscopy ◽  
Lamin A ◽  
Intermediate Filament Proteins ◽  
Sted Microscopy

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.

scholarly journals Expansion stimulated emission depletion microscopy (ExSTED)

2018 ◽  
Author(s):  
Mengfei Gao ◽  
Riccardo Maraspini ◽  
Oliver Beutel ◽  
Amin Zehtabian ◽  
Britta Eickholt ◽  
...  
Keyword(s):  
Excited State ◽  
Optical Resolution ◽  
Super Resolution ◽  
Stimulated Emission ◽  
High Fidelity ◽  
Sted Microscopy ◽  
Structural Details ◽  
Stimulated Emission Depletion ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

AbstractStimulated emission depletion (STED) microscopy is routinely used to resolve the ultra-structure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

scholarly journals STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

2021 ◽  
Author(s):  
Pablo Carravilla ◽  
Anindita Dasgupta ◽  
Gaukhar Zhurgenbayeva ◽  
Dmytro I. Danylchuk ◽  
Andrey S. Klymchenko ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Spatial Resolution ◽  
Super Resolution ◽  
Membrane Dynamics ◽  
Live Cell ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Temporal And Spatial ◽  
Polarity Sensitive

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high temporal and spatial resolution and long acquisition times, that also allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolution, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dyes is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

scholarly journals Transient Fluorescence Labeling: Low Affinity—High Benefits

2021 ◽  
Vol 22 (21) ◽  
pp. 11799
Author(s):  
Maxim M. Perfilov ◽  
Alexey S. Gavrikov ◽  
Konstantin A. Lukyanov ◽  
Alexander S. Mishin
Keyword(s):  
Chemical Properties ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Image Resolution ◽  
Stimulated Emission ◽  
Fluorescence Labeling ◽  
Cellular Functions ◽  
Affinity Labels ◽  
Physico Chemical ◽  
Pros And Cons

Fluorescent labeling is an established method for visualizing cellular structures and dynamics. The fundamental diffraction limit in image resolution was recently bypassed with the development of super-resolution microscopy. Notably, both localization microscopy and stimulated emission depletion (STED) microscopy impose tight restrictions on the physico-chemical properties of labels. One of them—the requirement for high photostability—can be satisfied by transiently interacting labels: a constant supply of transient labels from a medium replenishes the loss in the signal caused by photobleaching. Moreover, exchangeable tags are less likely to hinder the intrinsic dynamics and cellular functions of labeled molecules. Low-affinity labels may be used both for fixed and living cells in a range of nanoscopy modalities. Nevertheless, the design of optimal labeling and imaging protocols with these novel tags remains tricky. In this review, we highlight the pros and cons of a wide variety of transiently interacting labels. We further discuss the state of the art and future perspectives of low-affinity labeling methods.

Photobleaching Reduction in Modulated Super Resolution Microscopy

Microscopy ◽  
2020 ◽  
Author(s):  
Jafar H Ghithan ◽  
Jennifer M Noel ◽  
Thomas J Roussel ◽  
Maureen A McCall ◽  
Bruce W Alphenaar ◽  
...  
Keyword(s):  
Continuous Wave ◽  
Imaging Techniques ◽  
Fluorescence Emission ◽  
Super Resolution ◽  
Optical Power ◽  
Stimulated Emission ◽  
Laser Beams ◽  
Synchronous Detection ◽  
Major Drawback ◽  
Sted Microscopy

Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases of photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive-index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

STED microscopy—super-resolution bio-imaging utilizing a stimulated emission depletion

Microscopy ◽  
2015 ◽  
Vol 64 (4) ◽  
pp. 227-236 ◽  
Author(s):  
Kohei Otomo ◽  
Terumasa Hibi ◽  
Yuichi Kozawa ◽  
Tomomi Nemoto
Keyword(s):  
Super Resolution ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Bio Imaging ◽  
Stimulated Emission Depletion

scholarly journals Exploring the Release of Toxic Oligomers from α-Synuclein Fibrils with Antibodies and STED Microscopy

Life ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 431
Author(s):  
Alessandra Bigi ◽  
Emilio Ermini ◽  
Serene W. Chen ◽  
Roberta Cascella ◽  
Cristina Cecchi
Keyword(s):  
Conformational Changes ◽  
De Novo ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Phospholipid Bilayer ◽  
Sted Microscopy ◽  
Cell Dysfunction ◽  
Intrinsically Disordered ◽  
Neuronal Membranes

α-Synuclein (αS) is an intrinsically disordered and highly dynamic protein involved in dopamine release at presynaptic terminals. The abnormal aggregation of αS as mature fibrils into intraneuronal inclusion bodies is directly linked to Parkinson’s disease. Increasing experimental evidence suggests that soluble oligomers formed early during the aggregation process are the most cytotoxic forms of αS. This study investigated the uptake by neuronal cells of pathologically relevant αS oligomers and fibrils exploiting a range of conformation-sensitive antibodies, and the super-resolution stimulated emission depletion (STED) microscopy. We found that prefibrillar oligomers promptly penetrate neuronal membranes, thus resulting in cell dysfunction. By contrast, fibril docking to the phospholipid bilayer is accompanied by αS conformational changes with a progressive release of A11-reactive oligomers, which can enter into the neurons and trigger cell impairment. Our data provide important evidence on the role of αS fibrils as a source of harmful oligomers, which resemble the intermediate conformers formed de novo during aggregation, underling the dynamic and reversible nature of protein aggregates responsible for α-synucleinopathies.

scholarly journals A synergistic strategy to develop photostable and bright dyes with long Stokes shift for super-resolution microscopy

2021 ◽  
Author(s):  
gangwei jiang ◽  
Tian-Bing Ren ◽  
Elisa D’Este ◽  
mengyi xiong ◽  
Bin Xiong ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Stokes Shift ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Live Cells ◽  
Sted Microscopy ◽  
New Class ◽  
Two Photon ◽  
Cellular Environment ◽  
Super Resolution Microscopy

Abstract The quality and application of super-resolution fluorescence imaging greatly lie in the properties of fluorescent probes. However, conventional fluorophores in a cellular environment often suffer from low brightness, poor photostability, and short Stokes shift (< 30 nm). Here we report a synergistic strategy to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibronic structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability as well as Stokes shift. The new fluorophore BDQF-6 exhibits around twofold greater brightness (ε × Φ = 6.6 × 104 L·mol− 1·cm− 1) and Stokes shift (56 nm) than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, BDQF-6 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine- and JF608-based probes, known as photostable fluorophores for STED imaging. More BDQF-6 derivatives were developed next, allowing us to perform wash-free organelles (mitochondria and lysosome) staining and protein labeling with ultrahigh signal-to-noise ratios (up to 106 folds) in confocal and STED microscopy of live cells, or two-photon and 3D STED microscopy of fixed cells. Furthermore, the strategy was well generalized to different types of dyes (pyronin, rhodol, coumarin, and Boranil), offering a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.

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 Aberration correction for improving the image quality in STED microscopy using the genetic algorithm

Nanophotonics ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 1971-1980 ◽  
Author(s):  
Luwei Wang ◽  
Wei Yan ◽  
Runze Li ◽  
Xiaoyu Weng ◽  
Jia Zhang ◽  
...  
Keyword(s):  
Genetic Algorithm ◽  
Wave Front ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Aberration Correction ◽  
Sted Microscopy ◽  
Laser Wave ◽  
Resolution Imaging ◽  
Compensation Approach

AbstractWith a purely optical modulation of fluorescent behaviors, stimulated emission depletion (STED) microscopy allows for far-field imaging with a diffraction-unlimited resolution in theory. The performance of STED microscopy is affected by many factors, of which aberrations induced by the optical system and biological samples can distort the wave front of the depletion beam at the focal plane to greatly deteriorate the spatial resolution and the image contrast. Therefore, aberration correction is imperative for STED imaging, especially for imaging thick specimens. Here, we present a wave front compensation approach based on the genetic algorithm (GA) to restore the distorted laser wave front for improving the quality of STED images. After performing aberration correction on two types of zebrafish samples, the signal intensity and the imaging resolution of STED images were both improved, where the thicknesses were 24 μm and 100 μm in the zebrafish retina sample and the zebrafish embryo sample, respectively. The results showed that the GA-based wave front compensation approach has the capability of correction for both system-induced and sample-induced aberrations. The elimination of aberrations can prompt STED imaging in deep tissues; therefore, STED microscopy can be expected to play an increasingly important role in super-resolution imaging related to the scientific research in biological fields.

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.

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