scholarly journals LIVE-PAINT: Super-Resolution Microscopy Inside Live Cells Using Reversible Peptide-Protein Interactions

2020 ◽  
Author(s):  
Curran Oi ◽  
Zoe Gidden ◽  
Louise Holyoake ◽  
Owen Kantelberg ◽  
Simon Mochrie ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Fluorescent Imaging ◽  
Peptide Sequence ◽  
Live Cells ◽  
New Approach ◽  
Organic Fluorophore ◽  
Super Resolution Microscopy ◽  
Short Peptide Sequence

AbstractWe present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extend data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein.

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.

scholarly journals A step-by-step protocol for performing LIVE-PAINT super-resolution imaging of proteins in live cells using reversible peptide-protein interactions

2020 ◽  
Author(s):  
Curran Oi ◽  
Zoe Gidden ◽  
Louise Holyoake ◽  
Owen Kantelberg ◽  
Simon Mochrie ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Live Cells ◽  
Resolution Method ◽  
Resolution Imaging ◽  
Detailed Protocol ◽  
Direct Fusion

Abstract Super-resolution imaging of proteins inside live cells is a powerful tool for investigating protein behavior. We have developed a super-resolution method we call LIVE-PAINT, which uses reversible peptide-protein interactions to achieve super-resolution inside live cells. This method is particularly useful for studying proteins which do not tolerate large genetic fusions, such as direct fusion to a fluorescent protein. Here, we provide a detailed protocol for the use of LIVE-PAINT in S. cerevisiae.

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 LIVE-PAINT allows super-resolution microscopy inside living cells using reversible peptide-protein interactions

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Curran Oi ◽  
Zoe Gidden ◽  
Louise Holyoake ◽  
Owen Kantelberg ◽  
Simon Mochrie ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Super Resolution ◽  
Living Cells ◽  
Super Resolution Microscopy

scholarly journals A New Approach to Super-Resolution Microscopy

2015 ◽  
Vol 23 (3) ◽  
pp. 8-11
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Super Resolution ◽  
New Approach ◽  
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 Enhanced super-resolution microscopy by extreme value based emitter recovery

2018 ◽  
Author(s):  
Hongqiang Ma ◽  
Wei Jiang ◽  
Jianquan Xu ◽  
Yang Liu
Keyword(s):  
Super Resolution ◽  
Extreme Value ◽  
New Approach ◽  
Manual Adjustment ◽  
Imaging Characteristics ◽  
Nanoscale Imaging ◽  
Super Resolution Microscopy ◽  
Adjustment Of Parameters ◽  
Imagej Plugin

ABSTRACTSuper-resolution localization microscopy allows visualization of biological structure at nanoscale resolution. However, the presence of heterogeneous background can degrade the nanoscale resolution by tens of nanometers and introduce significant image artifacts. Here we develop a new approach, referred to as extreme value based emitter recovery (EVER), to accurately recover the distorted fluorescent emitters from heterogeneous background. Through numerical simulation and biological experiments, we demonstrate that EVER significantly improves the accuracy and fidelity of the reconstructed super-resolution image for a wide variety of imaging characteristics. EVER requires no manual adjustment of parameters and is implemented as an easy-to-use ImageJ plugin that can immediately enhance the quality of super-resolution images. Our method paves the way for accurate nanoscale imaging of samples with heterogeneous background fluorescence, such as thicker tissue and cells.

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 Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction

2018 ◽  
Author(s):  
Jakub Pospíšil ◽  
Tomáš Lukeš ◽  
Justin Bendesky ◽  
Karel Fliegel ◽  
Kathrin Spendier ◽  
...  
Keyword(s):  
High Resolution ◽  
Open Source ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Live Cells ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
Raw Data ◽  
Bayesian Image Reconstruction

AbstractBackgroundStructured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution).FindingsFive complete and freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods, and with newer Bayesian restoration approaches which we are developing.ConclusionVarious methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments is not typically published. Publicly available, high quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data was processed with SIMToolbox, an open source and freely available software solution for SIM.

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