Coordinate-Targeted and Coordinate-Stochastic Super-Resolution Microscopy with the Reversibly Switchable Fluorescent Protein Dreiklang

ChemPhysChem ◽  
2014 ◽  
Vol 15 (4) ◽  
pp. 756-762 ◽  
Author(s):  
Nickels A. Jensen ◽  
Johann G. Danzl ◽  
Katrin I. Willig ◽  
Flavie Lavoie-Cardinal ◽  
Tanja Brakemann ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Super Resolution ◽  
Super Resolution Microscopy

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>

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 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.

scholarly journals Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiwoong Kwon ◽  
Jong-Seok Park ◽  
Minsu Kang ◽  
Soobin Choi ◽  
Jumi Park ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Super Resolution ◽  
Live Cell ◽  
High Quality ◽  
Super Resolution Microscopy

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>

scholarly journals Electrostatic control of photoisomerization pathways in proteins

Science ◽  
2020 ◽  
Vol 367 (6473) ◽  
pp. 76-79 ◽  
Author(s):  
Matthew G. Romei ◽  
Chi-Yun Lin ◽  
Irimpan I. Mathews ◽  
Steven G. Boxer
Keyword(s):  
Protein Design ◽  
Fluorescence Quantum Yield ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Molecular Devices ◽  
Electrostatic Effects ◽  
Generalized Framework ◽  
Electrostatic Control ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

Rotation around a specific bond after photoexcitation is central to vision and emerging opportunities in optogenetics, super-resolution microscopy, and photoactive molecular devices. Competing roles for steric and electrostatic effects that govern bond-specific photoisomerization have been widely discussed, the latter originating from chromophore charge transfer upon excitation. We systematically altered the electrostatic properties of the green fluorescent protein chromophore in a photoswitchable variant, Dronpa2, using amber suppression to introduce electron-donating and electron-withdrawing groups to the phenolate ring. Through analysis of the absorption (color), fluorescence quantum yield, and energy barriers to ground- and excited-state isomerization, we evaluate the contributions of sterics and electrostatics quantitatively and demonstrate how electrostatic effects bias the pathway of chromophore photoisomerization, leading to a generalized framework to guide protein design.

scholarly journals Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein Tagging on Protein Assemblies

ACS Nano ◽  
2015 ◽  
Vol 9 (11) ◽  
pp. 11034-11041 ◽  
Author(s):  
Ingrid C. Vreja ◽  
Ivana Nikić ◽  
Fabian Göttfert ◽  
Mark Bates ◽  
Katharina Kröhnert ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Protein Assemblies ◽  
Super Resolution Microscopy ◽  
Protein Tagging

scholarly journals Fluorophore-labelled RNA aptamers to common protein tags as super-resolution imaging reagents.

2020 ◽  
Author(s):  
Juan Wang ◽  
Avtar Singh ◽  
Abdullah Ozer ◽  
Warren R Zipfel
Keyword(s):  
Fluorescent Protein ◽  
Super Resolution ◽  
Rna Aptamers ◽  
Cellular Structures ◽  
Intracellular Proteins ◽  
Resolution Imaging ◽  
Green Fluorescent ◽  
Protein Tags ◽  
Super Resolution Microscopy ◽  
Conventional Antibody

Developing labelling methods that densely and specifically label targeted cellular structures is critically important for centroid localization-based super-resolution microscopy. Being easy and inexpensive to produce in the laboratory and of relatively small size, RNA aptamers have potential as a substitute for conventional antibody labelling. By using aptamers selected against common protein tags - GFP (green fluorescent protein) in this case - we demonstrate labelling methods using dSTORM-compatible fluorophores for STORM and hybridizable imager strands for DNA-PAINT super-resolution optical imaging of any cellular proteins fused to the aptamer binding target. We show that we can label both extracellular and intracellular proteins for super-resolution imaging, and that the method in particular, offers some interesting advantages for live cell super-resolution imaging of plasma membrane proteins.

scholarly journals Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with L p-regularized image reconstruction

Microscopy ◽  
2018 ◽  
Vol 67 (2) ◽  
pp. 89-98
Author(s):  
Tetsuichi Wazawa ◽  
Yoshiyuki Arai ◽  
Yoshinobu Kawahara ◽  
Hiroki Takauchi ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Time Lapse ◽  
Live Cells ◽  
Polarization Angle ◽  
Microscopy Technique ◽  
Present Technique ◽  
Super Resolution Microscopy ◽  
Power Densities

Abstract Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102–106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70–80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.

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