Non-fluorescent Dye Research Articles

Drop-ANd-See: a simple, real-time, and noninvasive technique for assaying plasmodesmal permeability.

Gating of plasmodesmata (PD) is a highly dynamic cellular process spatiotemporally controlled by various physiological, developmental, and environmental conditions. Here, we describe a quantitative approach named Drop-ANd-See (DANS), which allows for a real-time, in situ assessment of plasmodesmal permeability in an array of comparative studies. The power of the DANS assay lies in its simplicity: a membrane-permeable, non-fluorescent dye is loaded onto the adaxial epidermis of an intact leaf; the absorbed dye is cleaved by cellular esterases and become fluorescent yet membrane-impermeable; this symplasmic form then diffuses via PD through the mesophyll and into the abaxial epidermis, where the extent of fluorescent dye spreading can be imaged and quantified by confocal microscopy as a measure of cell-to-cell permeability. By employing this DANS assay, rapid changes in PD permeability upon chemical, biological, or environmental treatments can be easily analyzed. Furthermore, PD permeability as a phenotype or a trait of interest can be evaluated using various genetic backgrounds or mutants. We provide hereby an easy-to-follow visual guide of the DANS assay using Arabidopsis plants as an example along with a description of the step-by-step protocol.

Lab-evaluation of applicability selected hydrogeological tracers in physical/chemical conditions

In this research, the effects of a number of physical/chemical factors such as pH, sunlight, temperature, and salinity, as well as adsorption in porous media on the applicability of selected groundwater tracers are evaluated. Tracers from different categories as fluorescent dye tracers (uranine, eosin, and rhodamine B), chemical salts (NaCl and KCl), and non-fluorescent dye tracer (KMnO4) have been tested. This research was conducted in the laboratory. The results show that uranine losses its florescence in acid environments, while in alkaline conditions its florescence increases. The results also show that due to photochemical decay, eosin is the most unstable tracer if subjected to sunlight. KMnO4 turns to brown under sunlight and high temperature conditions, and may lose its usefulness as a tracer. Results also confirm that the fluorescence intensity of rhodamine B decreases with increasing temperature and/or salinity. Uranine and eosin have high resistance against high temperature and salinity conditions. An important factor in ground water tracing is adsorption of tracer in porous media environment. Our research show that rhodamine B would easily adsorb to fine grain porous media, while uranine and eosin are of high resistance against adsorption; KMnO4 is adsorbed easily too.

An In vitro Assay to Quantify Nitrosative Component of Oxidative Stress.

Oxidative stress is a major contributing factor in a variety of neurodegenerative and vascular diseases. In vitro assessment of a major oxidant reactive nitrogen oxide species (RNOS) using dihydrorhodamine 123 (DHR 123) is a useful assay to quantify the reactive oxygen species in a cell. DHR 123, non-fluorescent laser dye freely penetrates the cell membrane and stains the mitochondria. Density of staining varies with the level of peroxynitrite (O=NOO-); as a result of interaction of superoxide anion (O2-) and nitric oxide (NO). The fluorescence is read using a spectrophotometer. Cells are seeded in 24 or 96- well plate and DHR 123 working solution is added after appropriate treatment. The fluorescence is read after 60 minutes of incubation at 485/528 nm with spectrophotometer. This assay is more sensitive and forms a stable end product than comparable assays and takes 90 minutes to complete.

Universal Dark Quencher Based on “Clicked” Spectrally Distinct Azo Dyes

The first synthesis of an heterotrifunctional molecular scaffold derived from the popular DABCYL azo dye quencher has been achieved. The sequential derviatization of this trivalent azobenzene derivative with two other nonfluorescent azo dyes (Black Hole Quencher BHQ-1 and BHQ-3) and through effective reactions from the "bioconjugation chemistry" repertoire has led to an universal dark quencher (UDQ). This "clicked" poly azo dye is able to turn off an array of fluorophores covering the UV/NIR (300-750 nm) spectral range.

(−)-Epicatechin regenerates the chlorinating activity of myeloperoxidase in vitro and in neutrophil granulocytes

The heme-containing enzyme myeloperoxidase (MPO) is mainly expressed in polymorphonuclear leukocytes (PMNs), the most abundant immune cell type in the blood. Accordingly, MPO is classically attributed to the innate immune response against pathogens. Yet, new results also show immune-regulatory functions of the halogenating MPO activity including the formation of anti-inflammatory mediators. In this work we tested the ability of the flavonoid (−)-epicatechin to regenerate this enzymatic activity both in vitro at the isolated MPO–H2O2–Cl− system and ex vivo in human PMNs. For all experiments the non-fluorescent dye aminophenyl fluorescein (APF) was used. Upon oxidation by the MPO, the halogenation product hypochlorous acid (HOCl) fluorescein is formed which can be detected e.g. by flow cytometry. The in vitro- and ex vivo-results concordantly show that (−)-epicatechin is a suitable substrate to overcome a compound II accumulation of MPO which was experimentally forced by applying excess hydrogen peroxide. Thereby concentration-dependent effects of the flavan-3-ol were found in both cases and confirmed the proposed mode of action of (−)-epicatechin. The results are in accordance with previous stopped-flow kinetic studies which showed a high reactivity of the polyphenol with MPO compound II. The obtained data may contribute to the explanation of the well-known health promoting effects of (−)-epicatechin. Moreover, the presented study provides new insights into the role of MPO during inflammation.

Surface-Enhanced Raman Spectroscopy-Based, Homogeneous, Multiplexed Immunoassay with Antibody-Fragments-Decorated Gold Nanoparticles

We report the development of a novel single-step, multiplexed, homogeneous immunoassay platform for sensitive detection of protein targets based on our realization of high surface-enhanced raman spectroscopy (SERS) signal enhancement by controlled assembly of SERS nanoparticles. An essential design of this platform is the use of gold nanoparticles or nanorods codecorated with specially reduced antibody half-fragments, nonfluorescent Raman-active dyes, and passivating proteins as the SERS nanoparticles. These nanoparticles offer a facile approach to accomplish orientational immobilization of antibodies, minimized interparticle distance, multicolor Raman fingerprint coding, low fluorescence background, as well as excellent biocompatibility and stability. Through sandwiched antibody-antigen interactions, controlled assembly of SERS nanoparticles is realized with a strong SERS signal achieved via plasmonic coupling, creating an immunoassay platform for rapid, sensitive, multiplexed quantification of proteins. This platform is demonstrated for reproducible quantification of three cytokines, interferon gamma, interleukin-2, and tumor necrosis factor alpha, with large signal-to-noise ratio. It is also successfully applied to multiplexed cytokine analysis for T cell secretion studies in complicated biological samples. The developed SERS immunoassay platform may create a simple but valuable tool for facilitating accurate validation and early detection of disease biomarkers as well as for point-of-care tests in clinical diagnostics.

Synthesis of diphenylamino-carbazole substituted BODIPY dyes and their photovoltaic performance in dye-sensitized solar cells

Non-alkylated BODIPY dye YHO-1 and hexa-alkylated BODIPY dye YHO-2, which have a diphenylamino-carbazole moiety as an electron-donating group at the 8-position on the BODIPY core and a carboxyhexyl group as an anchoring group on the carbazole ring, were designed and developed as photosensitizers for dye-sensitized solar cells (DSSCs). From the molecular structures of YHO-1 and YHO-2, when the two dyes were adsorbed on the TiO2 surface, it is assumed that the BODIPY core is located in close proximity to the TiO2 surface. The dye YHO-2 (Φf = 0.62) exhibits a significantly higher fluorescence quantum yield (Φf) than YHO-1 (Φf = 0.06). The short-circuit photocurrent density (Jsc) and solar energy-to-electricity conversion yield (η) for a DSSC based on YHO-2 are greater than those of YHO-1. This work demonstrates that fluorescent BODIPY dyes can inject electrons efficiently from the BODIPY core to the conduction band (CB) of the TiO2 electrode, but non-fluorescent BODIPY dyes result in lowering of photocurrent generation due to radiationless relaxation of the photoexcited dye.

Drug Sensitivity Assay of Xanthomonas citri subsp. citri Using REMA Plate Method

[Abstract] Resazurin Microtiter Assay (REMA) is a simple, rapid, reliable, sensitive, safe and cost-effective measurement of cell viability. Resazurin detects cell viability by converting from a nonfluorescent dye to the highly red fluorescent dye resorufin in response to chemical reduction of growth medium resulting from cell growth (Palomino et al., 2002). The REMA assay can be used as a fluorogenic oxidation-reduction indicator in a variety of cells, including bacteria, yeast and eukaryotes (Silva et al., 2013).

Bioconjugatable Azo‐Based Dark‐Quencher Dyes: Synthesis and Application to Protease‐Activatable Far‐Red Fluorescent Probes

We describe the efficient synthesis and one-step derivatization of novel, nonfluorescent azo dyes based on the Black Hole Quencher-3 (BHQ-3) scaffold. These dyes were equipped with various reactive and/or bioconjugatable groups (azido, α-iodoacetyl, ketone, terminal alkyne, vicinal diol). The azido derivative was found to be highly reactive in the context of copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions and allowed easy synthetic access to the first water-soluble (sulfonated derivative) and aldehyde-modified BHQ-3 dyes, the direct preparation of which failed by means of conventional azo-coupling reactions. The aldehyde- and α-iodoacetyl-containing fluorescence quenchers were readily conjugated to aminooxy- and cysteine-containing peptides by the formation of a stable oxime or thioether linkage, respectively. Further fluorescent labeling of the resultant peptide conjugates with red- or far-red-emitting rhodamine or cyanine dyes through sequential and/or one-pot bioconjugations, led to novel Förster resonance energy transfer (FRET) based probes suitable for the in vivo detection and imaging of urokinase plasminogen activator, a key protease in cancer invasion and metastasis.

Propidium iodide for making heterochromatin more evident in the C-banding technique

The detection of regions of heterochromatin has been the subject of intense investigation. We investigated an adaptation of the commonly used technique by replacing the nonfluorescent dye, Giemsa, by a fluorescent one, propidium iodide. This adaptation produces greater contrast of the heterochromatic bands in metaphase chromosomes and can be especially valuable when the organisms studied possess heterochromatin that is pale and difficult to visualize. We discuss the interactions of these two dyes with DNA and the excitation of the fluorescent dye when irradiated with ultraviolet light.

Rapid Dispatches Highlights

BioTechniquesVol. 52, No. 6 Open AccessRapid Dispatches HighlightsNathan BlowNathan BlowSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113875AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail 3-for-3Combinatorial RNA interference (RNAi) is an important approach for researchers who are studying highly mutable viruses such as HIV-1. The ability to express multiple siRNAs from a single genetic construct can be both an effective and efficient technique when studying gene expression, or developing gene therapy strategies directed against viruses. A challenge in engineering these constructs is dealing with the stable intramolecular hairpins associated with short hairpin RNAs which act as stops for DNA polymerase I. Reporting in Rapid Dispatches, Olga Kretova and her colleagues from the Englhardt Institute for Molecular Biology in Moscow, Russia, describe a single step method for the construction of cassettes that simultaneously express three different siRNAs. Their data clearly demonstrated specific and efficient knockdown of the target genes for each of the three siRNAs. The approach, which uses specifically designed oligos that overlap in their loop sequences and PCR to limit hairpin formation, should enable more effective and rapid combinatorial RNAi experiments in the future.Furry FluorescenceTransgenic animals have played a major role in expanding our understanding of biological processes and disease. Transgenic mammals incorporating fluorescent reports often require long periods of time to generate, and genotyping of these animals requires invasive sampling and lengthy protocols. But in a new article published at Rapid Dispatches, Wilfried Kues and colleagues at the Friedrich-Loeffler-Institut in Germany, describe a new, non-invasive approach to genotyping these transgenic animals by imaging hair samples using a fluorescence microscope. Not only were the authors able to determine if an animal expressed the fluorescence marker of interest by imaging the hair samples, but quantitative assessment of fluorescence was also possible and allowed for the rapid differentiation of hemizygous and homozygous status. With the ability to non-invasively sample transgenic mammals and then quickly obtain genotype status, this new technique should find considerable usage in transgenic labs.Venus-fluorescence of mouse hair samples, dotted line indicates measured area for intensity histogram.Electric DiatomsOur understanding of the basic biological process of some organisms has been hampered by our inability to genetically engineer certain species. One such example of this is the little diatom, an important primary producer in marine ecosystems that has proven difficult to genetically transform. While microprojectile bombardment approaches have allowed for the introduction of exogenous DNA into diatoms, these methods are costly and not common in labs around the world. But as Hong-Ye Li and colleagues report in Rapid Dispatches this month, the ability to transform diatoms may be possible in any molecular biology lab. It turns out that, under the right conditions and media environment, the commonly used transformation technique of electroporation can be employed to transform diatoms. Li et al. describe the media composition, as well as the electroporator settings, yielding the best transformation efficiencies with diatoms. The authors detail three different plasmids transfected into diatoms, ranging in size up to 6.4 kb, and demonstrate the establishment of an inducible selection marker. Taken together, the protocol described by Li et al. should enable the routine transformation of diatoms, paving the way for genetic engineering within this important marine organism.Multipurpose StainsAmyloids are protein aggregates that have been found to be associated with a number of neurological diseases such as Alzheimers and Creutzfeldt-Jakob disease. Researchers typically identify and study amyloids through the use of a specialized fluorescent dye called thioflavin-T that binds amyloids. Although a common method to study amyloids, thioflavin-T is not uniquely specific for amyloids, and other amyloid binding compounds that have been developed are either non-fluorescent or not available for research purposes. However, an interesting discovery reported in Rapid Dispatches by Steingrimur Stefansson and his colleagues at the Fuzbein Technology Institute in Rockville, Maryland, should greatly expand the amyloid staining toolkit. The authors found that a class of commercially available benzothiazole/benzoxazole containing fluorescent DNA intercalators can bind amyloid peptide and hamster prion. The compounds can act as acceptors in thioflavin-T fluorescence energy transfer experiments as well as a reporter group for binding studies with Congo Red (a non-fluorescent dye that also binds amyloids). Available in a range of fluorescence (500-750nm), these dyes should find use in the biochemical characterization of amyloids and as tools for in vitro screening of potential therapeutics.FiguresReferencesRelatedDetails Vol. 52, No. 6 Follow us on social media for the latest updates Metrics Downloaded 183 times History Published online 3 April 2018 Published in print June 2012 Information© 2012 Author(s)PDF download

Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors

The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g. EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes (‘activate the fluorogen’). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (β2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.

First evidence of epithelial transport in tardigrades: a comparative investigation of organic anion transport

We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotus crispae using a new method for quantifying non-fluorescent dyes. We compared the results acquired from the tardigrade with CPR transport data obtained from Malpighian tubules of the desert locust Schistocerca gregaria. CPR accumulated in the midgut lumen of H. crispae, indicating that organic anion transport takes place here. Our results show that CPR transport is inhibited by the mitochondrial un-coupler DNP (1 mmol l(-1); 81% reduction), the Na(+)/K(+)-ATPase inhibitor ouabain (10 mmol l(-1); 21% reduction) and the vacuolar H(+)-ATPase inhibitor bafilomycin (5 μmol l(-1); 21% reduction), and by the organic anions PAH (10 mmol l(-1); 44% reduction) and probenecid (10 mmol l(-1); 61% reduction, concentration-dependent inhibition). Transport by locust Malpighian tubules exhibits a similar pharmacological profile, albeit with markedly higher concentrations of CPR being reached in S. gregaria. Immunolocalization of the Na(+)/K(+)-ATPase α-subunit in S. gregaria revealed that this transporter is abundantly expressed and localized to the basal cell membranes. Immunolocalization data could not be obtained from H. crispae. Our results indicate that organic anion secretion by the tardigrade midgut is transporter mediated with likely candidates for the basolateral entry step being members of the Oat and/or Oatp transporter families. From our results, we cautiously suggest that apical H(+) and possibly basal Na(+)/K(+) pumps provide the driving force for the transport; the exact coupling between electrochemical gradients generated by the pumps and transport of ions, as well as the nature of the apical exit step, are unknown. This study is, to our knowledge, the first to show active epithelial transport in tardigrades.

Donnan Electric Potential Dependence of Intraparticle Diffusion of Malachite Green in Single Cation Exchange Resin Particles: A Laser Trapping-Microspectroscopy Study

A laser trapping-microspectroscopy technique combined with excitation energy transfer from a fluorescent cationic dye (Rhodamine B, RB+) to a non-fluorescent cationic dye (Malachite Green, MG+) was employed to study pH effects on the diffusion coefficients of MG+ (D(MG+)) in single cation-exchange resin microparticles with the diameters of 16 μm. When RB+-pre-adsorbed resin particles were soaked in an aqueous MG+ solution, the RB+ fluorescence was quenched gradually with the soaking time. The time course of the quenching efficiency of RB+ by MG+ was then used to evaluate the D(MG+) value in the particle. The D(MG+) value increased from 1.1 × 10-11 to 4.3 × 10-11 cm2.s–1 on going the solu- tion pH value from 9 to 4. The results were explained reasonably by a Donnan electric potential model.

Optoacoustic Imaging: An Emerging Modality for the Gastrointestinal Tract

Optoacoustic Imaging: An Emerging Modality for the Gastrointestinal Tract

Fluorescent labeling of proteins with amine-specific 1,3,2-(2H)-dioxaborine polymethine dye

A novel water-soluble amine-reactive dioxaborine trimethine dye was synthesized in a good yield and characterized. The potential of the dye as a specific reagent for protein labeling was demonstrated with bovine serum albumin and lysozyme. Its interaction with proteins was studied by fluorescence spectroscopy and gel electrophoresis. The covalent binding of this almost nonfluorescent dye to proteins results in a 75- to 78-fold increase of its emission intensity accompanied by a red shift of the fluorescence emission maximum by 27 to 45nm, with fluorescence wavelengths of labeled biomolecules being more than 600nm. The dye does not require activation for the labeling reaction and can be used in a variety of bioassay applications.

Detection of nonfluorescent molecules using differential interference contrast thermal lens microscope for extended nanochannel chromatography

An ultrasensitive absorbance detector, the differential interference contrast thermal lens microscope (DIC-TLM), was employed for a chromatography system using silica nanochannel. Recently, separation of ultrasmall volume sample has been strongly required for single-cell biological and chemical analysis. Previously, we have developed a chromatography system using nanochannels of ~100 nm scale (extended nanochannels) fabricated on a silica substrate. The extended nanochromatography realized highly efficient separation of samples <1 fL without packing materials. However, its detection method was limited to fluorescence method due to the small volume, and a new detector based on absorbance has been required. On the contrary, we have also developed DIC-TLM, a photothermal spectrometer based on absorption and thermal relaxation of sample for determination of concentration of nonfluorescent molecules in extended nanochannel. In this paper, we combined the extended nanochromatography and the DIC-TLM for separation and detection of nonfluorescent dyes. Particularly, basic performances of the DIC-TLM including quantitative performance and sensitivity were deliberated for injected samples of ~fL volume.

Tuning random lasers by engineered absorption

We achieve control over the emission wavelength of a random laser experimentally by adjusting the amount of absorption of emission light. The random laser consists of elastic scatterers in a high quantum efficiency gain medium to which a nonfluorescent dye is added. This nonfluorescent dye shifts the net gain curve and thereby the emission peak of the random laser. Mode competition effects are observed and explained. We interpret our results using a theoretical model that describes the spectral response of the system as a function of the effective cavity decay time and (re)absorption processes.

Carbon nanotubes as photoprotectors of organic dyes: reversible photoreaction instead of permanent photo-oxidation

In this paper we report that single-walled carbon nanotubes (SWNTs) can protect surface adsorbed Rhodamine B (RhB) molecules from permanent photo-oxidation via a reversible reaction. Upon strong light irradiation at 514 nm, the SWNT-adsorbed RhB molecules were switched to a non-fluorescent form, which looked like ordinary bleaching behavior. However, after staying without light for several hours the non-fluorescent dye species turned back to the original fluorescent form. This on/off switching can be considered as a reversible photobleaching process of the dye molecules. Other irreversible photochemical pathways of RhB were strongly prohibited due to the presence of SWNTs, providing the dye molecules with a high resistance against permanent photodegradation. By determining the maximum number of reconvertable RhB molecules per unit length of the nanotubes, we have further proved that this effect only works for the first layer of adsorbed dye molecules on the SWNT surfaces.

Photounbinding of Calmodulin from a Family of CaM Binding Peptides

BackgroundRecent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding.Methodology/Principal FindingsWe present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked.Conclusions/SignificanceThe experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner.