Dye selection for live cell imaging of intact siRNA

2012 ◽  
Vol 393 (1-2) ◽  
pp. 23-35 ◽  
Author(s):  
Markus Hirsch ◽  
Dennis Strand ◽  
Mark Helm
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Live Cell ◽  
High Yield ◽  
Ph Variation ◽  
Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

Advances in Microscopy Techniques

2011 ◽  
Vol 135 (2) ◽  
pp. 255-263
Author(s):  
Daniel B Schmolze ◽  
Clive Standley ◽  
Kevin E Fogarty ◽  
Andrew H Fischer
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Near Field ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Techniques ◽  
Live Cell ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Microscopy Techniques

Abstract Context.—Advances in microscopy enable visualization of a broad range of new morphologic features. Objective.—To review and illustrate advances in microscopy with relevance to pathologists. Data Sources.—Literature review and new observations. Results.—Fluorescence microscopy enables multiantigen detection; allows novel optical-sectioning techniques, with some advantages compared to paraffin sectioning; and permits live-cell imaging. Live-cell imaging allows pathologists to move from a period when all diagnostic expertise was reliant on interpreting static images to a period when cellular dynamics can play a role in diagnosis. New techniques have bypassed by about 100-fold what had long been believed to be a limit to the resolution of light microscopy. Fluorescence resonance energy transfer (FRET) appears capable of visualizing diagnostically relevant molecular events in living or fixed cells that are immeasurable by other molecular techniques. We describe applications of 2-photon microscopy, FRET, structured illumination, and the subdiffraction techniques of near-field microscopy, photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy. Conclusion.—New microscopy techniques present opportunities for pathologists to develop improved diagnostic tests.

Rhodamine-modified upconversion nanoprobe for distinguishing Cu2+ from Hg2+ and live cell imaging

2016 ◽  
Vol 40 (4) ◽  
pp. 3543-3551 ◽  
Author(s):  
Yanxia Xu ◽  
Huifang Li ◽  
Xianfu Meng ◽  
Jinliang Liu ◽  
Lining Sun ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Mesoporous Silica ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cell ◽  
Upconversion Nanoparticles ◽  
Inorganic Hybrid ◽  
Hybrid Nanoprobe

A new organic–inorganic hybrid nanoprobe based on luminescence resonance energy transfer (LRET) from mesoporous silica coated upconversion nanoparticles to a rhodamine B derivative was prepared for distinguishing Cu2+ from Hg2+ and live cell imaging applications.

scholarly journals Time-Lapse, Live-Cell Imaging of Potassium Efflux in Cell Exposed to Nanosecond Pulsed Electric Fields

2021 ◽  
Vol 120 (3) ◽  
pp. 223a
Author(s):  
Flavia Mazzarda ◽  
Esin B. Sozer ◽  
Julia L. Pittaluga ◽  
Claudia Muratori ◽  
P. Thomas Vernier
Keyword(s):  
Electric Fields ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Pulsed Electric Fields ◽  
Time Lapse ◽  
Live Cell ◽  
Potassium Efflux ◽  
Nanosecond Pulsed Electric Fields

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 Time-lapse live cell imaging to monitor doxorubicin release from DNA origami nanostructures

2018 ◽  
Vol 6 (11) ◽  
pp. 1605-1612 ◽  
Author(s):  
Yun Zeng ◽  
Jiajun Liu ◽  
Shuo Yang ◽  
Wenyan Liu ◽  
Liang Xu ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Dna Origami ◽  
Live Cell ◽  
Doxorubicin Release

DNA origami nanostructures can serve as a promising carrier for drug delivery due to the outstanding programmability and biocompatibility.

scholarly journals CRISPR/dual-FRET molecular beacon for sensitive live-cell imaging of non-repetitive genomic loci

2019 ◽  
Vol 47 (20) ◽  
pp. e131-e131 ◽  
Author(s):  
Shiqi Mao ◽  
Yachen Ying ◽  
Xiaotian Wu ◽  
Christopher J Krueger ◽  
Antony K Chen
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Molecular Beacon ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dynamic Imaging ◽  
Molecular Beacons ◽  
Imaging Systems ◽  
Guide Rna

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-based genomic imaging systems predominantly rely on fluorescent protein reporters, which lack the optical properties essential for sensitive dynamic imaging. Here, we modified the CRISPR single-guide RNA (sgRNA) to carry two distinct molecular beacons (MBs) that can undergo fluorescence resonance energy transfer (FRET) and demonstrated that the resulting system, CRISPR/dual-FRET MB, enables dynamic imaging of non-repetitive genomic loci with only three unique sgRNAs.

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