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 photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

Microscopy ◽  
2021 ◽  
Author(s):  
Tetsuichi Wazawa ◽  
Ryohei Noma ◽  
Shusaku Uto ◽  
Kazunori Sugiura ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ph Dependence ◽  
Super Resolution ◽  
Time Lapse ◽  
Light Irradiation ◽  
Acquisition Time ◽  
Polarization Angle ◽  
Cellular Dynamics

Abstract Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein

2020 ◽  
Vol 73 (8) ◽  
pp. 686
Author(s):  
Ashley M. Rozario ◽  
Fabian Zwettler ◽  
Sam Duwé ◽  
Riley B. Hargreaves ◽  
Aaron Brice ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Super Resolution ◽  
Time Lapse ◽  
Live Cells ◽  
Conventional Imaging ◽  
P Protein ◽  
Conventional Microscopy ◽  
P Proteins ◽  
Super Resolution Microscopy

The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

scholarly journals Meeting experiments at the diffraction barrier: an in-silico widefield fluorescence microscopy

2021 ◽  
Author(s):  
Subhamoy Mahajan ◽  
Tian Tang
Keyword(s):  
Fluorescence Microscopy ◽  
In Silico ◽  
Super Resolution ◽  
Light Sheet ◽  
Atomic Resolution ◽  
Live Cells ◽  
Two Photon ◽  
Color Combinations ◽  
Super Resolution Microscopy ◽  
Diffraction Barrier

AbstractFluorescence microscopy allows the visualization of live cells and their components, but even with advances in super- resolution microscopy, atomic resolution remains unattainable. On the other hand, molecular simulations (MS) can easily access atomic resolution, but comparison with experimental microscopy images has not been possible. In this work, a novel in-silico widefield fluorescence microscopy is proposed, which reduces the resolution of MS to generate images comparable to experiments. This technique will allow cross-validation and compound the knowledge gained from experiments and MS. We demonstrate that in-silico images can be produced with different optical axis, object focal planes, exposure time, color combinations, resolution, brightness and amount of out-of-focus fluorescence. This allows the generation of images that resemble those obtained from widefield, confocal, light-sheet, two-photon and super-resolution microscopy. This technique not only can be used as a standalone visualization tool for MS, but also lays the foundation for other in-silico microscopy methods.

Corrigendum to: ‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein

2020 ◽  
Vol 73 (8) ◽  
pp. 822
Author(s):  
Ashley M. Rozario ◽  
Fabian Zwettler ◽  
Sam Duwé ◽  
Riley B. Hargreaves ◽  
Aaron Brice ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Super Resolution ◽  
Time Lapse ◽  
Live Cells ◽  
Conventional Imaging ◽  
P Protein ◽  
Conventional Microscopy ◽  
P Proteins ◽  
Super Resolution Microscopy

The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

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.

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>

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 34-35
Author(s):  
Derek Toomre ◽  
Patrick Keller ◽  
Elena Diaz ◽  
Jamie White ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Video Microscopy ◽  
Internal Reflection ◽  
Fast Time ◽  
Cellular Polarity ◽  
Microscopy Technique

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

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 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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