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 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.

Long-term STED imaging of amyloid fibers with exchangeable Thioflavin T

Nanoscale ◽  
2020 ◽  
Vol 12 (28) ◽  
pp. 15050-15053
Author(s):  
Joaquim Torra ◽  
Patricia Bondia ◽  
Sylvia Gutierrez-Erlandsson ◽  
Begoña Sot ◽  
Cristina Flors
Keyword(s):  
Super Resolution ◽  
Stimulated Emission ◽  
Thioflavin T ◽  
Amyloid Fibers ◽  
Resolution Imaging ◽  
Stimulated Emission Depletion

We report the use of the amyloid probe Thioflavin T (ThT) as a specific and exchangeable fluorophore for stimulated emission depletion (STED) super-resolution imaging of amyloid fibers.

An indirectly stimulated emission depletion method for STED microscopy

2019 ◽  
Vol 52 (41) ◽  
pp. 415108
Author(s):  
Zhi-Jun Luo ◽  
Ya-Nan Liu ◽  
Meng-Lin Chen ◽  
Zong-Song Gan ◽  
Chang-Sheng Xie
Keyword(s):  
Stimulated Emission ◽  
Sted Microscopy ◽  
Stimulated Emission Depletion

scholarly journals Precision targeted ruthenium(ii) luminophores; highly effective probes for cell imaging by stimulated emission depletion (STED) microscopy

2016 ◽  
Vol 7 (10) ◽  
pp. 6551-6562 ◽  
Author(s):  
Aisling Byrne ◽  
Christopher S. Burke ◽  
Tia E. Keyes
Keyword(s):  
Cell Imaging ◽  
Stimulated Emission ◽  
Cell Organelles ◽  
Polypyridyl Complexes ◽  
Sted Microscopy ◽  
Highly Effective ◽  
Stimulated Emission Depletion ◽  
Peptide Targeting

Using precision peptide targeting to discrete cell organelles, it is demonstrated that Ru(ii) polypyridyl complexes are highly effective probes for stimulated emission depletion microscopy.

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 Shedding New Lights Into STED Microscopy: Emerging Nanoprobes for Imaging

2021 ◽  
Vol 9 ◽  
Author(s):  
Yanfeng Liu ◽  
Zheng Peng ◽  
Xiao Peng ◽  
Wei Yan ◽  
Zhigang Yang ◽  
...  
Keyword(s):  
Molecular Probes ◽  
Research Field ◽  
Stimulated Emission ◽  
Light Power ◽  
Sted Microscopy ◽  
Imaging Quality ◽  
Polymer Dots ◽  
Pros And Cons

First reported in 1994, stimulated emission depletion (STED) microscopy has long been regarded as a powerful tool for real-time superresolved bioimaging . However, high STED light power (101∼3 MW/cm2) is often required to achieve significant resolution improvement, which inevitably introduces phototoxicity and severe photobleaching, damaging the imaging quality, especially for long-term cases. Recently, the employment of nanoprobes (quantum dots, upconversion nanoparticles, carbon dots, polymer dots, AIE dots, etc.) in STED imaging has brought opportunities to overcoming such long-existing issues. These nanomaterials designed for STED imaging show not only lower STED power requirements but also more efficient photoluminescence (PL) and enhanced photostability than organic molecular probes. Herein, we review the recent progress in the development of nanoprobes for STED imaging, to highlight their potential in improving the long-term imaging quality of STED microscopy and broadening its application scope. We also discuss the pros and cons for specific classes of nanoprobes for STED bioimaging in detail to provide practical references for biological researchers seeking suitable imaging kits, promoting the development of relative research field.

scholarly journals EASY TWO-PHOTON IMAGE-SCANNING MICROSCOPY WITH SPAD ARRAY AND BLIND IMAGE RECONSTRUCTION

2019 ◽  
Author(s):  
S. V. Koho ◽  
E. Slenders ◽  
G. Tortarolo ◽  
M. Castello ◽  
M. Buttafava ◽  
...  
Keyword(s):  
Image Reconstruction ◽  
Single Photon ◽  
Imaging Modality ◽  
Scanning Microscopy ◽  
Array Detector ◽  
Reconstruction Method ◽  
Image Scanning ◽  
Two Photon ◽  
Blind Image

ABSTRACTTwo-photon excitation (2PE) microscopy is the imaging modality of choice, when one desires to work with thick biological samples, possibly in-vivo. However, the resolution in two-photon microscopy is poor, below confocal microscopy, and the lack of an optical pinhole becomes apparent in complex samples as reduced quality of optical sectioning. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced ISM platform – based on a new single-photon avalanche diode array detector – coupled with a novel blind image reconstruction method, is shown to improve the optical resolution, as well as the overall image quality in various test samples. Most importantly, our 2PE-ISM implementation requires no calibration or other input from the user – it works like any old and familiar two-photon system, but simply produces higher resolution images (in real-time). Making the complexity disappear, in our view, is the biggest novelty here, and the key for making 2PE-ISM mainstream.

Sign in / Sign up

Export Citation Format

Share Document