Fluorogenic Protein Probes with Red or Near-Infrared Emission for Genetically Targeted Live-Cell Imaging

2020 ◽  
Author(s):  
Sylvestre P. J. T. Bachollet ◽  
Cyril Addi ◽  
Jean-Maurice Mallet ◽  
Blaise Dumat
Keyword(s):  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Infrared Emission ◽  
Live Cell ◽  
Molecular Rotor ◽  
Model Protein ◽  
Near Infrared Emission

A series of red-emitting and near-infrared fluorogenic protein probes based on push-pull molecular rotor structures was developed. After characterization of their optical properties using Bovine Serum Albumin as a model protein, they were conjugated to a halogenoalkane ligand in order to target the protein self-labeling tag HaloTag. The interaction with HaloTag was investigated in vitro and then the most promising probes were applied to live-cell imaging in wash-free conditions using fluorogenic and chemogenetic targeting of HaloTag fusion proteins.<br>

scholarly journals Fluorogenic Protein Probes with Red or Near-Infrared Emission for Genetically Targeted Live-Cell Imaging

2020 ◽  
Author(s):  
Sylvestre P. J. T. Bachollet ◽  
Cyril Addi ◽  
Jean-Maurice Mallet ◽  
Blaise Dumat
Keyword(s):  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Infrared Emission ◽  
Live Cell ◽  
Molecular Rotor ◽  
Model Protein ◽  
Near Infrared Emission

A series of red-emitting and near-infrared fluorogenic protein probes based on push-pull molecular rotor structures was developed. After characterization of their optical properties using Bovine Serum Albumin as a model protein, they were conjugated to a halogenoalkane ligand in order to target the protein self-labeling tag HaloTag. The interaction with HaloTag was investigated in vitro and then the most promising probes were applied to live-cell imaging in wash-free conditions using fluorogenic and chemogenetic targeting of HaloTag fusion proteins.<br>

scholarly journals Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

2021 ◽  
Author(s):  
Keiichiro Sakai ◽  
Yohei Kondo ◽  
Hiroyoshi Fujioka ◽  
Mako Kamiya ◽  
Kazuhiro Aoki ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Infrared Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Yeast Cells ◽  
Color Palette

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with near-infrared excitation and emission maxima. Unlike the other conventional fluorescent proteins, iRFP requires biliverdin (BV) as a chromophore. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP-PCB was higher than that of iRFP-BV in vitro and in fission yeast. We introduced SynPCB, a PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids carrying the iRFP-fused marker proteins together with SynPCB. These tools not only enable the easy use of the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

scholarly journals Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

2021 ◽  
Author(s):  
Keiichiro Sakai ◽  
Yohei Kondo ◽  
Hiroyoshi Fujioka ◽  
Mako Kamiya ◽  
Kazuhiro Aoki ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Infrared Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Yeast Cells ◽  
Color Palette

Near-infrared fluorescent protein (iRFP) is the bright and stable fluorescent protein with excitation and emission maxima at 690 nm and 713 nm, respectively. Unlike the other conventional fluorescent proteins such as GFP, iRFP requires biliverdin (BV) as a chromophore because iRFP originates from phytochrome. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP attached to PCB was higher than that attached to BV in vitro and in fission yeast. We introduced SynPCB, a previously reported PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids, which contain genes required for the SynPCB system and the iRFP-fused marker proteins. These tools not only enable the easy use of iRFP in fission yeast and the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the doors to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

scholarly journals Uniform Spheres of α-NaYF4:RE3+ (RE=Eu, Tb, Ce, Er, and Tm): Template-Free Synthesis, Multi-Color Photoluminescence, and Their Application in Cellular Imaging

Crystals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 119
Author(s):  
Xiaofeng Fan ◽  
Laiqin Gu ◽  
Yiling Hu ◽  
Qi Zhu
Keyword(s):  
Near Infrared ◽  
Infrared Emission ◽  
Particle Sizes ◽  
Hydrothermal Processing ◽  
High Brightness ◽  
Luminescent Probes ◽  
Bio Imaging ◽  
Template Free ◽  
Near Infrared Emission

Uniformly dispersed luminescent probes with a high brightness and high resolution are desired in bio imaging fields. Here, ~100 nm sized and well-dispersed spheres of RE3+ doped α-NaYF4 (rare earth (RE) = Eu, Tb, Ce, Er, and Tm) have been facile synthesized through hydrothermal processing in the absence of a template, followed by a proper annealing. The processing window of the cubic structured spheres is wide, because the hydrothermal products are independent of the processing conditions, including reaction time and temperature. The original morphology and crystal structure can be well retained with a calcination temperature up to 600 °C. However, calcination gives rise to a reduction of particle sizes, as a result of the crystallite growth and densification. Under ultraviolet radiation, α-NaYF4:RE3+ spheres show characteristic f-f emissions of RE3+ (RE = Eu, Tb, Ce, Er, and Tm), and exhibit orange red, green, ultraviolet (UV), blue green, and blue emissions, respectively. Mainly because of the near-infrared emission at ~697 nm (5D0→7F4 transitions of Eu3+), the successful imaging of macrophages was achieved by NH2-NaYF4:Eu3+ probes, indicating their excellent imaging capacity for cells in vitro.

scholarly journals Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

ACS Nano ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. 302-315 ◽  
Author(s):  
Eric Alonas ◽  
Aaron W. Lifland ◽  
Manasa Gudheti ◽  
Daryll Vanover ◽  
Jeenah Jung ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Virus Dynamics ◽  
In Cells

scholarly journals A Near‐Infrared Photoswitchable Protein–Fluorophore Tag for No‐Wash Live Cell Imaging

2018 ◽  
Vol 130 (49) ◽  
pp. 16315-16319 ◽  
Author(s):  
Wei Sheng ◽  
Setare Tahmasebi Nick ◽  
Elizabeth M. Santos ◽  
Xinliang Ding ◽  
Jun Zhang ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Live Cell

A new visible-light-excitable ICT-CHEF-mediated fluorescence ‘turn-on’ probe for the selective detection of Cd2+ in a mixed aqueous system with live-cell imaging

2015 ◽  
Vol 44 (12) ◽  
pp. 5763-5770 ◽  
Author(s):  
Shyamaprosad Goswami ◽  
Krishnendu Aich ◽  
Sangita Das ◽  
Chitrangada Das Mukhopadhyay ◽  
Deblina Sarkar ◽  
...  
Keyword(s):  
Visible Light ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Aqueous System ◽  
Live Cell ◽  
Selective Detection ◽  
Turn On ◽  
Fluorescence Turn On

A new quinoline based sensor was developed and applied for the selective detection of Cd2+ both in vitro and in vivo.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies

2021 ◽  
Vol 22 (20) ◽  
pp. 11092
Author(s):  
Magalie Bénard ◽  
Damien Schapman ◽  
Christophe Chamot ◽  
Fatéméh Dubois ◽  
Guénaëlle Levallet ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Light Exposure ◽  
Photon Counting ◽  
Live Cell ◽  
Stimulated Emission ◽  
Plot Analysis ◽  
Phasor Plot

Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.

scholarly journals A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

2020 ◽  
Author(s):  
Felix Pahmeier ◽  
Christoper J Neufeldt ◽  
Berati Cerikan ◽  
Vibhu Prasad ◽  
Costantin Pape ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Reporter System ◽  
Infected Cells ◽  
Positive Strand Rna

ABSTRACTPositive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCEReporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

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