Green and Red Fluorescent Dyes for Translational Applications in Imaging and Sensing Analytes: A Dual‐Color Flag

Oliveira, Elisabete; Bértolo, Emilia; Núñez, Cristina; Pilla, Viviane; Santos, Hugo M.; Fernández‐Lodeiro, Javier; Fernández-Lodeiro, Adrián; Djafari, Jamila; Capelo-Martínez, José Luis; Lodeiro, Carlos

doi:10.1002/open.201700177

Cited by 17 publications

(13 citation statements)

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“…On nFCM, AF488 and PE performed equally well and surprisingly, allowed for increased sEV staining efficiencies over the red fluorophores AF647 and APC, even though AF488 theoretically should have the lowest brightness (extinction coefficient x quantum yield) out of them all (see the Supplementary Table S1 ). Generally, red fluorophores may also be more prone to self-quenching, consequently diminishing their quantum yield [ 51 ]; therefore, it would be relevant to address the properties of such dyes within the scope of single nanoparticle analysis. For F-NTA, AF488 provided the optimal balance between stability and brightness.…”

Section: Discussionmentioning

confidence: 99%

Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

Fortunato¹,

Mladenović

Criscuoli³

et al. 2021

IJMS

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The relevance of extracellular vesicles (EVs) has grown exponentially, together with innovative basic research branches that feed medical and bioengineering applications. Such attraction has been fostered by the biological roles of EVs, as they carry biomolecules from any cell type to trigger systemic paracrine signaling or to dispose metabolism products. To fulfill their roles, EVs are transported through circulating biofluids, which can be exploited for the administration of therapeutic nanostructures or collected to intercept relevant EV-contained biomarkers. Despite their potential, EVs are ubiquitous and considerably heterogeneous. Therefore, it is fundamental to profile and identify subpopulations of interest. In this study, we optimized EV-labeling protocols on two different high-resolution single-particle platforms, the NanoFCM NanoAnalyzer (nFCM) and Particle Metrix ZetaView Fluorescence Nanoparticle Tracking Analyzer (F-NTA). In addition to the information obtained by particles’ scattered light, purified and non-purified EVs from different cell sources were fluorescently stained with combinations of specific dyes and antibodies to facilitate their identification and characterization. Despite the validity and compatibility of EV-labeling strategies, they should be optimized for each platform. Since EVs can be easily confounded with similar-sized nanoparticles, it is imperative to control instrument settings and the specificity of staining protocols in order to conduct a rigorous and informative analysis.

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Section: Discussionmentioning

confidence: 99%

Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

Fortunato¹,

Mladenović

Criscuoli³

et al. 2021

IJMS

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“…Some of the most used fluorophores in histology are fluorescein isothiocyanate (FITC; green/yellow), TRITC (red), rhodamine (red), Texas red (red), cyanine 2 (Cy2; green), indocarbocyanine (Cy3; red) and aminomethyl coumarin (AMCA; blue) [118]. Red and green fluorescent dyes are broadly applied due to their sensitivity and fast responses in optical imaging systems [137]. In this sense, fluorescein and rhodamine can be broadly used to evaluate cells, tissues and whole animals [138], considering their excellent optical properties due to the long excitation wavelength and high fluorescence quantum yield [137].…”

Section: Fluorescence Techniquesmentioning

confidence: 99%

‘A picture is worth a thousand words’: The use of microscopy for imaging neuroinflammation

Fraga

Tassinari

Jantsch

et al. 2021

Clinical and Experimental Immunology

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Since the first studies of the nervous system by the Nobel laureates Camillo Golgi and Santiago Ramon y Cajal using simple dyes and conventional light microscopes, microscopy has come a long way to the most recent techniques that make it possible to perform images in live cells and animals in health and disease. Many pathological conditions of the central nervous system have already been linked to inflammatory responses. In this scenario, several available markers and techniques can help imaging and unveil the neuroinflammatory process. Moreover, microscopy imaging How to cite this article: de Fraga LS, Tassinari ID, Jantsch J, Guedes RP, Bambini-Junior V. 'A picture is worth a thousand words': The use of microscopy for imaging neuroinflammation.

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“…1,2 This is indeed also the case in the field of bio-imaging and for assays relying on fluorescent dyes, where enhanced water solubility of dyes leads to improved signal to background ratios and elimination of artifacts. [3][4][5] Organic chromophores in general consist of extended π-conjugated systems responsible for absorption and emission of ultra-violet (UV) to near-infrared (NIR) electromagnetic radiation.…”

Section: Introductionmentioning

confidence: 99%

What Is Best Strategy for Water Soluble Fluorescence Dyes? – a Case Study Using Long Fluorescence Lifetime DAOTA Dyes

Bisballe

Laursen

2020

Preprint

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The applications of organic fluorophores in biological sciences rely heavily on their properties in aqueous solution. The lipophilic nature of virtually all such chromophores provides several challenges to adapt them to biologically relevant conditions. In this work we investigate three different strategies for achieving water-solubility of the diazaoxatriangulenium (DAOTA+) chromophore: hydrophilic counter ions, aromatic sulfonation of the chromophore core, and attachment of cationic or zwitterionic side chains. The long fluorescence lifetime (FLT, τf » 20 ns) of DAOTA+ makes it a sensitive probe for changes in the rate of non-radiative deactivation and for aggregation leading to multi exponential decay profiles. Direct sulfonation of the chromophore, as applied in several Alexa dyes, does indeed increase solubility drastically, but at the cost of greatly reduced quantum yields (QY) due to enhanced non-radiative deactivation processes. The introduction of either cationic (4) or zwitterionic side chains (5), however, brings the FLT (τf = 18 ns) and QY (φf = 0.56) of the dye to the same level as the parent chromophore in acetonitrile. For these derivatives time-resolved fluorescence spectroscopy also reveals a high resistance to aggregation and non-specific binding in a high loading of bovine serum albumin (BSA). The results clearly show that addition of charged flexible side chains is preferable to direct sulfonation of the chromophore core.

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Green and Red Fluorescent Dyes for Translational Applications in Imaging and Sensing Analytes: A Dual‐Color Flag

Cited by 17 publications

References 0 publications

Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

‘A picture is worth a thousand words’: The use of microscopy for imaging neuroinflammation

What Is Best Strategy for Water Soluble Fluorescence Dyes? – a Case Study Using Long Fluorescence Lifetime DAOTA Dyes

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