Live-Cell STED Microscopy with Genetically Encoded Biosensor

Nano Letters ◽  
2015 ◽  
Vol 15 (5) ◽  
pp. 2928-2932 ◽  
Author(s):  
Natalia M. Mishina ◽  
Alexander S. Mishin ◽  
Yury Belyaev ◽  
Ekaterina A. Bogdanova ◽  
Sergey Lukyanov ◽  
...  
Keyword(s):  
Live Cell ◽  
Sted Microscopy ◽  
Genetically Encoded Biosensor

Three dimensional live-cell STED microscopy at increased depth using a water immersion objective

2018 ◽  
Vol 89 (5) ◽  
pp. 053701 ◽  
Author(s):  
Jörn Heine ◽  
Christian A. Wurm ◽  
Jan Keller-Findeisen ◽  
Andreas Schönle ◽  
Benjamin Harke ◽  
...  
Keyword(s):  
Water Immersion ◽  
Three Dimensional ◽  
Live Cell ◽  
Sted Microscopy ◽  
Water Immersion Objective

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 Synthesis of Fluorescent Jasplakinolide Analogues for Live-Cell STED Microscopy of Actin

2020 ◽  
Vol 85 (11) ◽  
pp. 7267-7275
Author(s):  
Vladimir N. Belov ◽  
Stefan Stoldt ◽  
Franziska Rüttger ◽  
Michael John ◽  
Jan Seikowski ◽  
...  
Keyword(s):  
Live Cell ◽  
Sted Microscopy

scholarly journals Rhodamines with a chloronicotinic acid fragment for live cell superresolution STED** microscopy

2021 ◽  
Author(s):  
Vladimir N Belov ◽  
Florian Grimm ◽  
Jasmin Rehman ◽  
Stefan Stoldt ◽  
Taukeer A. Khan ◽  
...  
Keyword(s):  
Live Cell ◽  
Sted Microscopy ◽  
Acid Fragment

scholarly journals Bleaching-independent, whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores

2018 ◽  
Author(s):  
Christoph Spahn ◽  
Jonathan B. Grimm ◽  
Luke D. Lavis ◽  
Marko Lampe ◽  
Mike Heilemann
Keyword(s):  
Super Resolution ◽  
Living Cells ◽  
Live Cell ◽  
Acquisition Time ◽  
Fluorescence Signal ◽  
Target Structure ◽  
Whole Cell ◽  
Sted Microscopy ◽  
Resolution Imaging ◽  
Constant Exchange

AbstractWe demonstrate bleaching-independent STED microscopy using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging in fixed and living cells. Foremost, we achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy with up to 100 min acquisition time.

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 Live-cell imaging of dendritic spines by STED microscopy

2008 ◽  
Vol 105 (48) ◽  
pp. 18982-18987 ◽  
Author(s):  
U. V. Nagerl ◽  
K. I. Willig ◽  
B. Hein ◽  
S. W. Hell ◽  
T. Bonhoeffer
Keyword(s):  
Dendritic Spines ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Sted Microscopy

scholarly journals In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Waja Wegner ◽  
Peter Ilgen ◽  
Carola Gregor ◽  
Joris van Dort ◽  
Alexander C. Mott ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Live Cell ◽  
Sted Microscopy

scholarly journals Synergic Combination of Stimulated Emission Depletion Microscopy with Image Scanning Microscopy to Reduce Light Dosage

2019 ◽  
Author(s):  
Giorgio Tortarolo ◽  
Marco Castello ◽  
Sami Koho ◽  
Giuseppe Vicidomini
Keyword(s):  
Single Photon ◽  
Simulated Data ◽  
Live Cell ◽  
Stimulated Emission ◽  
Beam Intensity ◽  
Scanning Microscopy ◽  
Image Scanning ◽  
Sted Microscopy ◽  
Stimulated Emission Depletion

AbstractStimulated emission depletion (STED) microscopy is one of the most influential nanoscopy techniques; by increasing the STED beam intensity, it theoretically improves the spatial resolution to any desired value. However, the higher is the dose of stimulating photons, the stronger are the photo-bleaching and photo-toxicity effects, which potentially compromise live-cell and long-term imaging. For this reason the scientific community is looking for strategies to reduce the STED beam intensity needed to achieve a target resolution. Here, we show how the combination of STED microscopy with image scanning microscopy (ISM) meets this request. In particular, we introduce a new STED-ISM architecture – based on our recent single-photon-avalanche-diode (SPAD) detector array – which allows covering the near-diffraction limit resolution range with reduced STED beam intensity. We demonstrate this ability both with simulated data and in live-cell experiments. Because of (i) the minimal changes in the optical architecture of the typical point-scanning STED microscope; (ii) the parameter-free, robust and real-time pixel-reassignment method to obtain the STED-ISM image; (iii) the compatibility with all the recent progresses in STED microscopy, we envisage a natural and rapid upgrade of any STED microscope to the proposed STED-ISM architecture.

scholarly journals Fast STED Microscopy for Live Cell Imaging

2011 ◽  
Vol 100 (3) ◽  
pp. 355a
Author(s):  
Marcel A. Lauterbach ◽  
Volker Westphal ◽  
Dirk Kamin ◽  
Chaitanya K. Ullal ◽  
Silvio O. Rizzoli ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Sted Microscopy
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