Characterization of Photophysical Properties of Photoactivatable Fluorescent Proteins for Super-Resolution Microscopy

2020 ◽  
Vol 124 (10) ◽  
pp. 1892-1897
Author(s):  
Antai Tao ◽  
Rongjing Zhang ◽  
Junhua Yuan
Keyword(s):  
Fluorescent Proteins ◽  
Photophysical Properties ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Fluorescent Probes for STED Optical Nanoscopy

Nanomaterials ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 21
Author(s):  
Sejoo Jeong ◽  
Jerker Widengren ◽  
Jong-Chan Lee
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Photophysical Properties ◽  
Optical Resolution ◽  
Super Resolution ◽  
Fluorescent Nanoparticles ◽  
Resolution Limit ◽  
Photochemical Properties ◽  
Super Resolution Microscopy

Progress in developing fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, is inseparable from the advancement in optical fluorescence microscopy. Super-resolution microscopy, or optical nanoscopy, overcame the far-field optical resolution limit, known as Abbe’s diffraction limit, by taking advantage of the photophysical properties of fluorescent probes. Therefore, fluorescent probes for super-resolution microscopy should meet the new requirements in the probes’ photophysical and photochemical properties. STED optical nanoscopy achieves super-resolution by depleting excited fluorophores at the periphery of an excitation laser beam using a depletion beam with a hollow core. An ideal fluorescent probe for STED nanoscopy must meet specific photophysical and photochemical properties, including high photostability, depletability at the depletion wavelength, low adverse excitability, and biocompatibility. This review introduces the requirements of fluorescent probes for STED nanoscopy and discusses the recent progress in the development of fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, for the STED nanoscopy. The strengths and the limitations of the fluorescent probes are analyzed in detail.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<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>

Fluorescent Proteins and Super-Resolution Microscopy

2017 ◽  
Vol 37 (3) ◽  
pp. 0318008
Author(s):  
彭鼎铭 Peng Dingming ◽  
付志飞 Fu Zhifei ◽  
徐平勇 Xu Pingyong
Keyword(s):  
Fluorescent Proteins ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy

2017 ◽  
Vol 56 (22) ◽  
pp. 6131-6135 ◽  
Author(s):  
Anne Burgert ◽  
Jan Schlegel ◽  
Jérôme Bécam ◽  
Sören Doose ◽  
Erhard Bieberich ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Super Resolution ◽  
Super Resolution Microscopy

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

ACS Nano ◽  
2015 ◽  
Vol 9 (10) ◽  
pp. 9528-9541 ◽  
Author(s):  
Sam Duwé ◽  
Elke De Zitter ◽  
Vincent Gielen ◽  
Benjamien Moeyaert ◽  
Wim Vandenberg ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

scholarly journals Can Super-Resolution Microscopy Become a Standard Characterization Technique for Material Chemistry?

2021 ◽  
Author(s):  
Shikha Dhiman ◽  
Teodora Andrian ◽  
Beatriz Santiago ◽  
Marrit Tholen ◽  
Yuyang Wang ◽  
...  
Keyword(s):  
Super Resolution ◽  
Analytical Techniques ◽  
Material Chemistry ◽  
Characterization Technique ◽  
Super Resolution Microscopy ◽  
Synthesized Materials

The characterization of newly synthesized materials is a cornerstone of all chemistry and nanotechnology laboratories. For this purpose, a wide array of analytical techniques have been standardized and are used...

scholarly journals Embracing the uncertainty: the evolution of SOFI into a diverse family of fluctuation-based super-resolution microscopy methods

2021 ◽  
Author(s):  
Monika Pawlowska ◽  
Ron Tenne ◽  
Bohnishikha Ghosh ◽  
Adrian Makowski ◽  
Radek Lapkiewicz
Keyword(s):  
3D Imaging ◽  
Deep Neural Networks ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Significant Progress ◽  
Spatial Correlations ◽  
Order Of Magnitude ◽  
Temporal And Spatial ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

Abstract Super-resolution microscopy techniques have pushed the limits of resolution in optical imaging by more than an order of magnitude. However, these methods often require long acquisition times as well as complex setups and sample preparation protocols. Super-resolution Optical Fluctuation Imaging (SOFI) emerged over ten years ago as an approach that exploits temporal and spatial correlations within the acquired images to obtain increased resolution with less strict requirements. This review follows the progress of SOFI from its first demonstration to the development of a branch of methods that treat fluctuations as a source of contrast, rather than noise. Among others, we highlight the implementation of SOFI with standard fluorescent proteins as well as the microscope modification that facilitate 3D imaging and the application of modern cameras. Going beyond the classical framework of SOFI, we explore different innovative concepts from deep neural networks all the way to a quantum analogue of SOFI, antibunching microscopy. While SOFI has not reached the same level of ubiquity as other super-resolution methods, our overview finds significant progress and substantial potential for the concept of leveraging fluorescence fluctuations to obtain super-resolved images.

scholarly journals Application of SNAP-Tag in Expansion Super-Resolution Microscopy Using DNA Oligostrands

2021 ◽  
Vol 9 ◽  
Author(s):  
Longfang Yao ◽  
Li Zhang ◽  
Yiyan Fei ◽  
Liwen Chen ◽  
Lan Mi ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
New Technology ◽  
Super Resolution ◽  
Target Protein ◽  
Substrate Molecule ◽  
Label System ◽  
Actual Target ◽  
Super Resolution Microscopy

Expansion super-resolution technology is a new technology developed in recent years. It anchors the dye on the hydrogel and the dye expands with the expansion of the hydrogel so that a super-resolution map can be obtained under an ordinary microscope. However, by labeling the target protein with a first antibody and secondary antibody, the distance between the fluorescent group and the actual target protein is greatly increased. Although fluorescent proteins can also be used for expansion super-resolution to reduce this effect, the fluorescent protein is often destroyed during sample preparation. To solve this problem, we developed a novel label system for expansion microscopy, based on a DNA oligostrand linked with a fluorescent dye, acrylamide group (linker), and benzoylguanine (BG, a small substrate molecule for SNAP-tag). This protocol greatly reduced the error between the position of fluorescent group and the actual target protein, and also reduced loss of the fluorescent group during sample preparation.

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