scholarly journals Design of a protein tag and fluorogenic probe with modular structure for live-cell imaging of intracellular proteins

2016 ◽  
Vol 7 (1) ◽  
pp. 308-314 ◽  
Author(s):  
Yuko Kamikawa ◽  
Yuichiro Hori ◽  
Kazuo Yamashita ◽  
Lin Jin ◽  
Shinya Hirayama ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Modular Structure ◽  
Live Cells ◽  
Fluorogenic Probe ◽  
Intracellular Proteins ◽  
Protein Tag ◽  
Modular Structures

Quick and no-wash labeling of intracellular proteins was achieved in live cells using a PYP-tag mutant and a membrane-permeable fluorogenic probe with modular structures.

scholarly journals Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

2017 ◽  
Author(s):  
Noa Aloush ◽  
Tomer Schvartz ◽  
Andres I. König ◽  
Sarit Cohen ◽  
Eugene Brozgol ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Stop Codon ◽  
Signal To Noise Ratio ◽  
Trna Synthetase ◽  
Live Cell ◽  
Live Cells ◽  
Copy Numbers ◽  
Intracellular Proteins

ABSTRACTGenetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine synthetase/ pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNA contributes to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cells images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

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 CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

2020 ◽  
Author(s):  
Patricia A. Clow ◽  
Nathaniel Jillette ◽  
Jacqueline J. Zhu ◽  
Albert W. Cheng
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Repetitive Sequences ◽  
Binary Sequence ◽  
Three Dimensional ◽  
Live Cell ◽  
Live Cells ◽  
Imaging Method ◽  
Genomic Locus ◽  
Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

scholarly journals Live Cell Imaging: 3-Dimensional Tracking of Non-blinking ‘Giant’ Quantum Dots in Live Cells (Adv. Funct. Mater. 30/2014)

2014 ◽  
Vol 24 (30) ◽  
pp. 4795-4795 ◽  
Author(s):  
Aaron M. Keller ◽  
Yagnaseni Ghosh ◽  
Matthew S. DeVore ◽  
Mary E. Phipps ◽  
Michael H. Stewart ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Live Cells ◽  
3 Dimensional

scholarly journals The Cation−π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification

Biochemistry ◽  
2017 ◽  
Vol 56 (11) ◽  
pp. 1585-1595 ◽  
Author(s):  
Yu Liu ◽  
Kun Miao ◽  
Noah P. Dunham ◽  
Hongbin Liu ◽  
Matthew Fares ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Protein Quantification ◽  
Live Cell ◽  
Free Protein ◽  
Fluorogenic Probe

scholarly journals Label-free and live cell imaging by interferometric scattering microscopy

2018 ◽  
Vol 9 (10) ◽  
pp. 2690-2697 ◽  
Author(s):  
Jin-Sung Park ◽  
Il-Buem Lee ◽  
Hyeon-Min Moon ◽  
Jong-Hyeon Joo ◽  
Kyoung-Hoon Kim ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Complex Structure ◽  
Live Cell ◽  
Fluorescent Label ◽  
Live Cells ◽  
Label Free ◽  
Cytoplasmic Organelles ◽  
Microscopic Techniques

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label.

A Rapid SNAP-Tag Fluorogenic Probe Based on an Environment-Sensitive Fluorophore for No-Wash Live Cell Imaging

2014 ◽  
Vol 9 (10) ◽  
pp. 2359-2365 ◽  
Author(s):  
Tao-Kai Liu ◽  
Pei-Ying Hsieh ◽  
Yu-De Zhuang ◽  
Chi-Yang Hsia ◽  
Chi-Ling Huang ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Fluorogenic Probe

scholarly journals PinMol: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs

2018 ◽  
Author(s):  
Livia V. Bayer ◽  
Omar S. Omar ◽  
Diana P. Bratu ◽  
Irina E. Catrina
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Molecular Beacon ◽  
Structural Information ◽  
Live Cell ◽  
Molecular Beacons ◽  
Intramolecular Interactions ◽  
Live Cells ◽  
Target Rna

ABSTRACTMolecular beacons are nucleic acid oligomers labeled with a fluorophore and a quencher that fold in a hairpin-shaped structure, which fluoresce only when bound to their target RNA. They are used for the visualization of endogenous mRNAs in live cells. Here, we report a Python program (PinMol) that designs molecular beacons best suited for live cell imaging by using structural information from secondary structures of the target RNA, predicted via energy minimization approaches. PinMol takes into account the accessibility of the targeted regions, as well as the inter- and intramolecular interactions of each selected probe. To demonstrate its applicability, we synthesized an oskar mRNA-specific molecular beacon (osk1236), which is selected by PinMol to target a more accessible region than a manually designed oskar-specific molecular beacon (osk2216). We previously demonstrated osk2216 to be efficient in detecting oskar mRNA in in vivo experiments. Here, we show that osk1236 outperformed osk2216 in live cell imaging experiments.

Light up ClO−in live cells using an aza-coumarin based fluorescent probe with fast response and high sensitivity

The Analyst ◽  
2015 ◽  
Vol 140 (13) ◽  
pp. 4594-4598 ◽  
Author(s):  
Jiangli Fan ◽  
Huiying Mu ◽  
Hao Zhu ◽  
Jingyun Wang ◽  
Xiaojun Peng
Keyword(s):  
Fluorescent Probe ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
High Sensitivity ◽  
Fast Response ◽  
Live Cell ◽  
Live Cells

An aza-coumarin based fluorescent and colorimetricAC-ClOfor the ClO−determination with fast response and high sensitivity.AC-ClOwas successfully applied for the live-cell imaging of exogenous and endogenous ClO−.

scholarly journals HaloTag8: an engineered protein tag to improve fluorophore performance

2021 ◽  
Author(s):  
Michelle S. Frei ◽  
Miroslaw Tarnawski ◽  
Julia Roberti ◽  
Birgit Koch ◽  
Julien Hiblot ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Probes ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Protein Tag

HaloTag8 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag8 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag8 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging.

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