Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Phosphine quenching of cyanine dyes as a versatile tool for fluorescence microscopy.

ABSTRACT: We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with UV light dissociates the adduct and returns the dye to the fluorescent state. We demonstrate that TCEP quenching can be used for super-resolution imaging as well as for other applications, such as differentiating between molecules inside and outside the cell.

SUBMITTER: Vaughan JC

PROVIDER: S-EPMC3624894 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Phosphine quenching of cyanine dyes as a versatile tool for fluorescence microscopy.

Vaughan Joshua C JC Dempsey Graham T GT Sun Eileen E Zhuang Xiaowei X

Journal of the American Chemical Society 20130117 4

We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with UV light dissociates the adduct and returns the dye to the fluorescent state. We demonstrate that TCEP quenching can be used for super-resolution imaging as well as for o ...[more]

PMID: 23311875

Similar Datasets

Sequence-dependent fluorescence of cyanine dyes on microarrays.

Project description:Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.

| S-EPMC3143128 | biostudies-literature

"Turn-on" protein fluorescence: in situ formation of cyanine dyes.

Project description:Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2-11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues.

| S-EPMC4311949 | biostudies-other

Sterically Shielded Heptamethine Cyanine Dyes for Bioconjugation and High Performance Near-Infrared Fluorescence Imaging.

Project description:The near-infrared window of fluorescent heptamethine cyanine dyes greatly facilitates biological imaging because there is deep penetration of the light and negligible background fluorescence. However, dye instability, aggregation, and poor pharmacokinetics are current drawbacks that limit performance and the scope of possible applications. All these limitations are simultaneously overcome with a new molecular design strategy that produces a charge balanced and sterically shielded fluorochrome. The key design feature is a meso-aryl group that simultaneously projects two shielding arms directly over each face of a linear heptamethine polyene. Cell and mouse imaging experiments compared a shielded heptamethine cyanine dye (and several peptide and antibody bioconjugates) to benchmark heptamethine dyes and found that the shielded systems possess an unsurpassed combination of photophysical, physiochemical, and biodistribution properties that greatly enhance bioimaging performance.

| S-EPMC7473488 | biostudies-literature

Photoswitching mechanism of cyanine dyes.

Project description:Cyanine dyes have been shown to undergo reversible photoswitching, where the fluorophore can be switched between a fluorescent state and a dark state upon illumination at different wavelengths. The photochemical mechanism by which switching occurs has yet to be elucidated. In this study, we have determined the mechanism of photoswitching by characterizing the kinetics of dark state formation and the spectral and structural properties of the dark state. The rate of switching to the dark state depends on the concentration of the primary thiol in the solution and the solution pH in a manner quantitatively consistent with the formation of an encounter complex between the cyanine dye and ionized thiol prior to their conjugation. Mass spectrometry suggests that the photoconversion product is a thiol-cyanine adduct in which covalent attachment of the thiol to the polymethine bridge disrupts the original conjugated pi-electron system of the dye.

| S-EPMC2797371 | biostudies-literature

Heterobifunctional Dyes: Highly Fluorescent Linkers Based on Cyanine Dyes.

Project description:Herein, we present a new synthetic route to cyanine-based heterobifunctional dyes and their application as fluorescent linkers between polymers and biomolecules. The synthesized compounds, designed in the visible spectral range, are equipped with two different reactive groups for highly selective conjugation under physiological conditions. By applying indolenine precursors with functionalized benzenes, we achieved water-soluble asymmetric cyanine dyes bearing maleimido and N-hydroxysuccinimidyl functionalities in a three-step synthesis. Spectroscopic characterization revealed good molar absorption coefficients and moderate fluorescence quantum yields. Further reaction with polyethylene glycol yielded dye-polymer conjugates that were subsequently coupled to the antibody cetuximab, often applied in cancer therapy. Successful coupling was confirmed by mass shifts detected by gel electrophoresis. Receptor-binding studies and live-cell imaging revealed that labeling did not alter the biological function. In sum, we provided a successful synthetic pathway to rigid heterobifunctional cyanine dyes that are applicable as fluorescent linkers, for example, for connecting antibodies with macromolecules. Our approach contributes to the field of bioconjugation chemistry, such as antibody-drug conjugates by combining diagnostic and therapeutic approaches.

| S-EPMC5474662 | biostudies-literature

Fluorescence quenching: A tool for single-molecule protein-folding study.

Project description:By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. In the native folded state, the fluorescence from dye molecules is quenched due to the close proximity between the dye molecules. Unfolding of the titin leads to a dramatic increase in the fluorescence intensity. Such a change makes the folded and unfolded states of a single titin molecule clearly distinguishable and allows us to measure the folding dynamics of individual titin molecules in real time. We have also shown that fluorescence quenching can signal folding and unfolding of a small protein with only one immunoglobulin domain.

| S-EPMC18902 | biostudies-literature

Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy.

Project description:Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent "ON" state. This helps in identifying structures of interest. Subsequently, labelled samples are induced into blinking, which is necessary for determining the position of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor 647 (AF647), blinks in Vectashield. However, after preparing immunocytochemical samples in Vectashield, we noticed that the fluorescence intensity of AF647 is quenched. This is particularly evident for dimmer immunostainings, such as stainings of some components of neuronal cytoskeleton and axonal initial segment. Because structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) a quantitative analysis of the suitability of Vectashield for dSTORM imaging of high and low-abundance AF647-labelled targets. Furthermore, for the first time, we quantitatively analyse the performance of Alexa Fluor Plus 647, a new variant of AF647-conjugated antibody, in dSTORM imaging.

| S-EPMC7160131 | biostudies-literature

Fluorescence quenching of (dimethylamino)naphthalene dyes Badan and Prodan by tryptophan in cytochromes P450 and micelles.

Project description:Fluorescence of 2-(N,N-dimethylamino)-6-propionylnaphthalene dyes Badan and Prodan is quenched by tryptophan in Brij 58 micelles as well as in two cytochrome P450 proteins (CYP102, CYP119) with Badan covalently attached to a cysteine residue. Formation of nonemissive complexes between a dye molecule and tryptophan accounts for about 76% of the fluorescence intensity quenching in micelles, the rest is due to diffusive encounters. In the absence of tryptophan, fluorescence of Badan-labeled cytochromes decays with triexponential kinetics characterized by lifetimes of about 100 ps, 700-800 ps, and 3 ns. Site mutation of a histidine residue in the vicinity of the Badan label by tryptophan results in shortening of all three decay lifetimes. The relative amplitude of the fastest component increases at the expense of the two slower ones. The average quenching rate constants are 4.5 × 10(8) s(-1) (CYP102) and 3.7 × 10(8) s(-1) (CYP119), at 288 K. Cyclic voltammetry of Prodan in MeCN shows a reversible reduction peak at -1.85 V vs NHE that becomes chemically irreversible and shifts positively upon addition of water. A quasireversible reduction at -0.88 V was observed in an aqueous buffer (pH 7.3). The excited-state reduction potential of Prodan (and Badan) is estimated to vary from about +0.6 V (vs NHE) in polar aprotic media (MeCN) to approximately +1.6 V in water. Tryptophan quenching of Badan/Prodan fluorescence in CYPs and Brij 58 micelles is exergonic by ?0.5 V and involves tryptophan oxidation by excited Badan/Prodan, coupled with a fast reaction between the reduced dye and water. Photoreduction is a new quenching mechanism for 2-(N,N-dimethylamino)-6-propionylnaphthalene dyes that are often used as solvatochromic polarity probes, FRET donors and acceptors, as well as reporters of solvation dynamics.

| S-EPMC4148165 | biostudies-literature

A new structure-activity relationship for cyanine dyes to improve photostability and fluorescence properties for live cell imaging.

Project description:A new set of cyanine-indole dyes was synthesized, characterized by optical and cytotoxic properties and subsequently applied for live cell imaging. Furthermore, these dyes were postsynthetically linked covalently to the 2'-position of uridine anchors in presynthesized oligonucleotides using the copper(i)-catalyzed azide-alkyne cycloaddition in order to evaluate their photostability and imaging properties in living cells. The nucleophilicity at position C-2 of the indole part of the dyes was elucidated as key for a new structure-activity relationship that served as a rational guide to improve the photostability and optical properties of these green-emitting dyes for live cell imaging of nucleic acids. While the photostability rises exponentially with decreasing nucleophilicity, thermal bleaching experiments confirmed an opposite trend supposing that the superoxide radical anion is mainly responsible for the photobleaching of the dyes. Furthermore, the cytotoxicities of the dyes were tested in HeLa cells and moderate to low LD50 values were obtained. This interdisciplinary strategy allowed us to identify one dye with excellent optical properties and even better photostability and decreased cytotoxicity compared to a cyanine-indole dye that bears an additional cyclooctatetraene group as a triplet state quencher.

| S-EPMC6115680 | biostudies-literature

TestSTORM: Versatile simulator software for multimodal super-resolution localization fluorescence microscopy.

Project description:Optimization of sample, imaging and data processing parameters is an essential task in localization based super-resolution microscopy, where the final image quality strongly depends on the imaging of single isolated fluorescent molecules. A computational solution that uses a simulator software for the generation of test data stacks was proposed, developed and tested. The implemented advanced physical models such as scalar and vector based point spread functions, polarization sensitive detection, drift, spectral crosstalk, structured background etc., made the simulation results more realistic and helped us interpret the final super-resolved images and distinguish between real structures and imaging artefacts.

| S-EPMC5430448 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data