Visible Dye Research Articles

Stain efficiency and MALDI-TOF MS compatibility of seven visible staining procedures

Visible stain is still the most popular protein staining method used in proteomic approaches. However, most published data have been derived from comparisons between visible dyes and fluorescent dyes. In this work, we have focused on seven widely used visible staining procedures--Neuhoff CCB, blue silver, and five silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN)--and studied their stain efficiencies and MALDI-TOF MS compatibilities on 1-D and 2-D PAGE. It was concluded that blue silver is slightly better in terms of stain efficiency than Neuhoff CCB, but it presented worse MS compatibility. Neuhoff CCB presented better MS compatibility and superior linearity but worse sensitivity than silver stains. Among the five silvering procedures, He SN showed the best MS compatibility and a reasonable staining efficiency; Yan SN lowered the chances of obtaining the protein identity by PMF but gave the best stain efficiency; Vorum SN gave a very clear background and a great contrast, while Blum SN was the worst in this respect. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.

Liposome-based immunostrip for the rapid detection of Salmonella

Salmonellae are ubiquitous human pathogens, which pose a danger to the elderly and children. Due to the increased number of outbreaks of human illness associated with the consumption of contaminated products in the USA and many other countries, there is an urgent need to develop rapid assays to detect common food-borne pathogens. This study demonstrates the feasibility of using a detectable label comprising methyl blue (MB), a visible dye, entrapped inside liposomes. Immunoliposomes tagged with anti-Salmonella common structural antigens (CSA) antibody encapsulating MB dye were prepared and used as the signal amplifier for the development of a field-portable colorimetric immunoassay to detect Salmonellae. Tapping mode atomic force microscopy (TMAFM), a scanning probe technique, was utilized to demonstrate the presence of anti-Salmonella antibody at the thus-prepared liposome. A plastic-backed nitrocellulose strip with two immobilized zones formed the basis of a sandwich assay. The first zone was the antigen capture zone (AC zone), used in a sandwich (noncompetitive) assay format; the other was the biotin capture zone (BC zone), used as a quality control index for the strip assay. During the capillary migration of the wicking reagent containing 80μL of immunoliposomes and 40μL of the test sample (heat-killed S. typhimurium), sample pathogens with surface-bound immunoliposomes were captured at the AC zone, while the unbound immunoliposomes continued to migrate and bind to the anti-biotin antibodies coated on the BC zone. The color density of the AC zone was directly proportional to the number of Salmonella typhimurium in the test sample. The detection limit of the current assay with heat-killed Salmonella typhimurium was 1,680 cells. The cross-reactivity of the proposed immunoassay was also investigated, and pathogens including E. coli O157:H7 and Listeria genus specific caused no interference with the detection of Salmonella typhimurium.

Comparative in vitro antimicrobial procedural efficacy for susceptibility of Staphylococcus aureus, Escherichia coli and Pseudomonas species to chloramphenicol, ciprofloxacin and cefaclor

The present study assesses the reliability of the in vitro susceptibility tests E-test, disc diffusion and Alamar blue-based microbroth dilution assays for evaluating the efficacy of chloramphenicol, ciprofloxacin and cefaclor against clinical and reference isolates of Staphylococcus aureus, Escherichia coli and Pseudomonas spp. for use in empirical therapy. Ciprofloxacin showed 82% agreement between E-test and microbroth dilution by the visible dye reduction method against all organisms. This figure was 67% for cefaclor and 60% for chloramphenicol. E-test and microbroth dilution showed excellent correlation against S. aureus with all three antibiotics; however, correlation was not observed in Pseudomonas spp. between E-test and microbroth dilution by the percentage dye reduction method. E. coli did not show significant correlation, indicating the presence of heteroresistance. Chloramphenicol, being bacteristatic in nature, did not show clear agreement between the susceptibility test methods used. This study indicates that E-test provides an indication of minimum inhibitory concentration (MIC) of a panel of antibiotics against clinical isolates; however, microbroth dilution (dye reduction) is the most sensitive method for the determination of MIC due to intracellular enzymatic reduction of the dye to formazan, which only occurs in viable organisms. The study highlights the need for harmonisation between E-test and microbroth dilution methods in clinical trials of new antibiotics and in monitoring the drug resistance patterns in community and healthcare settings.

Resonance-enhanced multiphoton ionization of jet-cooled 2-methylfuran

Mass-resolved, resonance-enhanced multiphoton ionization (REMPI) spectra of jet-cooled 2-methylfuran have been measured in various spectroscopic regions (visible and UV ns dye laser). The results have been interpreted as two-photon resonances and as three-photon resonances to the 3d Rydberg manifold chiefly. The 3d 1 A 1 ← ( X ∼ ) A 1 transition, located at 56,360 cm −1, is the strongest one in the (2 + 1) REMPI spectrum and has a discrete vibrational structure. The vibrational analysis has been performed. The three-photon resonance spectrum tracks the 3d VUV absorption spectrum. The strongest transition in this case is the 3d 1 B 2 ← ( X ∼ ) A 1 transition located at 56,013 cm −1. The transition to the 3s A 2 Rydberg state has been detected at 44,018 cm −1 (3 + 2 REMPI spectrum).

Processing of transparent materials using visible nanosecond laser pulses

Laser micromachining of transparent materials is an intensively studied research area from the point of view of microoptical element fabrication. One of the most promising indirect processing methods is the laser-induced back-side dry etching (LIBDE). During this method, transparent targets are contacted with solid thin layers, which absorb and transform the pulse energy resulting in etching. The applicability of LIBDE technology for processing of fused silica using a visible nanosecond dye laser (λ=500 nm, FWHM=11 ns) and a 100-nm-thick aluminium absorbing layer was investigated. The applied fluence was varied in the range of 0–3050 mJ/cm2; the illuminated area was 0.1 mm2. The threshold fluence of the LIBDE etching of fused silica was found to be approximately 540 mJ/cm2. The chemical composition of the surface layers on and around the etched holes was investigated by field-emission scanning electron microscopy and energy-dispersive X-ray spectrometry. It was found that on average 0.4±0.3 at. % aluminium is built into the upper ∼1-μm-thick volume of the illuminated fused silica, while the aluminium content fell below the detection limit in the case of the original surface. Our experiments proved that the LIBDE procedure is suitable for microprocessing of transparent materials using visible nanosecond laser light.

Comparison of visible and near-infrared wavelength-excitable fluorescent dyes for molecular imaging of cancer

Targeted fluorescent molecular imaging probes may provide an optimal means of detecting disease. Stable, organic fluorophores can be repeatedly excited in vivo by propagated light and consequentially can provide large signal-to-noise ratios (SNRs) for image detection of target tissues. In the literature, many small animal imaging studies are performed with a red excitable dye, Cy5.5, conjugated to the targeting component. We report the comparison of the in vivo fluorescent imaging performance of a near-IR (NIR) and a red-excitable dye. Epidermal growth factor (EGF) was conjugated with Cy5.5 [excitation/emission (ex/em), 660710 nm] or IRDye 800CW (ex/em: 785830 nm) for imaging EGF receptor (EGFr) positive (MDA-MB-468) and/or negative (MDA-MB-435) human breast cancer cell lines in subcutaneous xenograft models. The conjugates were injected intravenously at 1-nmol-dye equivalent with and without anti-EGFr monoclonal antibody C225, preadministered 24 h prior as a competitive ligand to EGFr. Our images show that while both agents target EGFr, the EGF-IRDye 800CW evidenced a significantly reduced background and enhanced the tumor-to-background ratio (TBR) compared to the EGF-Cy5.5. Immunohistochemistry shows that EGF causes activation of the EGFr signaling pathway, suggesting that prior to use as a targeting, diagnostic agent, potential deleterious effects should be considered.

Counterion‐dye staining for DNA in electrophoresed gels using indoine blue and methyl orange

In this study, we describe a sensitive staining method for DNA in agarose and polyacrylamide gels using organic visible dyes, indoine blue (IB) and methyl orange (MO). The counterion-dye staining method uses two oppositely charged dyes to form a hydrophobic ion pair complex in the staining solution. A decrease in the number of free forms of dyes in staining solution can enhance the selectivity of binding between the dye and DNA, and can reduce nonspecific background staining. As a result, the sensitivity of counterion-dye staining was significantly improved compared with other dye-based staining. This method uses a staining solution consisting of 0.008% IB, 0.002% MO, 10% ethanol and 0.2 M sodium acetate at pH 4.7, and can detect 5 ng of lambda DNA/HindIII within 60 min in agarose gels and 10 ng of PhiX174 DNA/HaeIII within 20 min in polyacrylamide gels.

Different Impact of Staining Procedures Using Visible Stains and Fluorescent Dyes for Large-Scale Investigation of Proteomes by MALDI-TOF Mass Spectrometry

Replicate 2-D gels were stained with four visible or fluorescent dyes using published procedures, and 48 co-detected spots were selected for contrasting values in abundance, M(r) and pI. Success rate of identification and sequence coverage were affected in a dye-dependent manner by the three parameters. Frequency of missed cleavages and recovery of sulfur-containing peptides also depended on the dye. Finally, the dataset was used to predict the number of proteins identifiable when integrating the differential contribution of each parameter. Sypro Ruby appeared to combine several favorable features: no dependence of the identification rate upon the physicochemical properties of proteins, no impact on frequency of missed cleavages, and a higher predicted identification rate.

Confocal imaging of [Ca2+] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence

Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca2+ from cellular stores into the cytosol, a process that appears to be tightly controlled by changes in [Ca2+] within the store. A method to image free [Ca2+] within cellular organelles was devised, which provided the first quantitative confocal images of [Ca2+] inside the sarcoplasmic reticulum (SR) of skeletal muscle. The method exploits, for greater sensitivity, the dual spectral shifts that some fluorescent dyes undergo upon binding Ca2+. It was implemented with mag-indo-1 trapped in the intracellular organelles of frog skeletal muscle and validated showing that it largely monitors [Ca2+] in a caffeine-sensitive compartment with the structure of the SR cisternae. A tentative calibration in situ demonstrated an increase in the dye's dissociation constant, not unlike that observed for other dyes in cellular environments. This increase, together with other characteristics of the ratioing method, placed the half-signal [Ca2+] near 1 mM, a value suitable for cellular stores. Demonstrated advantages of the technique include accuracy (that of a calibrated ratiometric method), dynamic range and sensitivity (from the combination of two spectral shifts), spatial and temporal resolution, and compatibility with a vast array of visible dyes to monitor diverse aspects of cellular function. SEER (shifted excitation and emission ratioing) also provides a [Ca2+]-independent measure of dye concentration in the cell. Store and mitochondrial [Ca2+] ([Ca2+]SR and [Ca2+]mito could be measured separately using the high spatial resolution of SEER. Evolution of [Ca2+]SR was followed upon changes in cytosolic [Ca2+] ([Ca2+]cyto). At [Ca2+]cyto = 100 nM, [Ca2+]mito remained near the lower limit of detection and [Ca2+]SR stabilized at values that were submillimolar according to our tentative calibration. Steady [Ca2+]SR was only slightly higher in 800 nM [Ca2+]cyto, and essentially did not decrease unless [Ca2+]cyto was reduced below 10 nM. While the increase of [Ca2+]SR was limited by loss through Ca2+ release channels, its decrease in low [Ca2+]cyto was largely dependent on leaks through the SR Ca2+ pump.

Rewritable phosphorescent paper by the control of competing kinetic and thermodynamic self-assembling events

Security inks have become of increasing importance. They are composed of invisible substances that provide printed images that are not able to be photocopied, and are readable only under special environments. Here we report a novel photoluminescent ink for rewritable media that dichroically emits phosphorescence due to a structural bistability of the self-assembled luminophor. Long-lasting images have been developed by using conventional thermal printers, which are readable only on exposure to ultraviolet light, and more importantly, are thermally erasable for rewriting. Although thermally rewritable printing media have already been developed using visible dyes and cholesteric liquid crystals, security inks that allow rewriting of invisible printed images are unprecedented. We realized this unique feature by the control of kinetic and thermodynamic processes that compete with one another in the self-assembly of the luminophor. This strategy can provide an important step towards the next-generation security technology for information handling.

The Assignment of the Different Infrared Continuum Absorbance Changes Observed in the 3000–1800-cm −1 Region during the Bacteriorhodopsin Photocycle

The bleach continuum in the 1900–1800-cm −1 region was reported during the photocycle of bacteriorhodopsin (bR) and was assigned to the dissociation of a polarizable proton chain during the proton release step. More recently, a broad band pass filter was used and additional infrared continua have been reported: a bleach at >2700 cm −1, a bleach in the 2500–2150-cm −1 region, and an absorptive behavior in the 2100–1800-cm −1 region. To fully understand the importance of the hydrogen-bonded chains in the mechanism of the proton transport in bR, a detailed study is carried out here. Comparisons are made between the time-resolved Fourier transform infrared spectroscopy experiments on wild-type bR and its E204Q mutant (which has no early proton release), and between the changes in the continua observed in thermally or photothermally heated water (using visible light-absorbing dye) and those observed during the photocycle. The results strongly suggest that, except for the weak bleach in the 1900–1800-cm −1 region and >2500 cm −1, there are other infrared continua observed during the bR photocycle, which are inseparable from the changes in the absorption of the solvent water molecules that are photothermally excited via the nonradiative relaxation of the photoexcited retinal chromophore. A possible structure of the hydrogen-bonded system, giving rise to the observed bleach in the 1900–1800-cm −1 region and the role of the polarizable proton in the proton transport is discussed.

New Products

New Products

Usefulness of visible dyes for the staining of protein or DNA in electrophoresis.

Since 1993, we have studied visible organic dye stains for protein or DNA to improve methodologies and developed the counterion dye staining method. The method employs two oppositely charged dyes that form an ion-pair complex in the staining solution. The selective binding of free dye to protein or DNA in the staining solution improves detection sensitivity and speed. It is a rapid and sensitive procedure, involving fixing/staining or staining/quick destaining steps that are completed in 1-1.5 h. The lowest detection limits achieved are 4-8 ng of protein on polyacrylamide gels and approximately 10 ng of DNA on agarose gels. The focus of this review is to chronicle the development and current status of the counterion dye staining method for detection of protein or DNA. As an extended application of visible dyes, we also discuss the visible dye staining method for detecting protein on blotting membranes developed in our laboratory.

Staining of Intraocular Lenses with Various Dyes: A Study of Digital Image Analysis

Purpose: To evaluate the interaction of various intraocular lens (IOL) materials with commonly used dyes. Materials and Methods: One IOL of each of the five groups (polymethylmethacrylate, silicone, three-piece hydrophobic acrylic, single-piece hydrophobic acrylic and single-piece hydrophilic acrylic) was bathed in trypan blue 0.1%, fluorescein sodium 2% and indocyanine green (ICG) 0.5% for 15 min. Digital photographs of the IOLs were obtained prior to bathing and after the 15-min bath in each dye. The same IOLs were immersed in the same dye for another 15 min and digital images were reobtained to depict the 30-min dye uptake. New IOLs from the group that exhibited visible colour change after the 15-min bath were then bathed in twofold dilution and if there was still visible dye uptake, fourfold dilution was performed, repeating the 15-min bath and digital photography. The images were then processed using Adobe Photoshop 5.1 to get mean luminosity and red-green-blue values. These values were compared between the groups and the undyed control. Also, dye washout was observed in serum-containing vials. Results: None of the polymethylmethacrylate, silicone and hydrophobic acrylic IOLs were stained with the dyes used. The only IOL material that changed colour was hydrophilic acrylic and did so with all dyes. The most marked colour change was with fluorescein sodium 2%, the least was with trypan blue 0.05%. Twofold dilution of trypan blue and fourfold dilutions of fluorescein sodium and ICG still stained the hydrophilic acrylic IOL; however, fourfold dilution of trypan blue did not cause a significant colour change. Trypan blue washed out within 6 h, while the IOL stained with ICG remained stained for longer than 24 h. Comment: Hydrophilic acrylic IOLs should be used with caution together with dyes since this material demonstrates marked dye uptake and washout may also take some time.

Proteomic capacity of recent fluorescent dyes for protein staining

Staining of two-dimensional gel constitutes a crucial step in comparative proteome analysis with respect to both the number of proteins analysed, the accuracy of spot quantification and reproducibility. In this work, we compared the efficiency of recent fluorophores to stain Arabidopsis total protein extract: Sypro Ruby ® (SR), Deep Purple ® (DP) and 5-hexadecanoylamino-fluorescein (C16-F). In addition, classical visible dyes, colloidal Coomassie blue (CCB) and silver nitrate (SN), were also included. High quality images were obtained for the three fluorescent dyes, DP giving the cleaner background, whereas spikes were observed with SR and a rough background with C16-F. On the other hand, saturation occurred for abundant spots with SR and DP. For a same protein load the number of detected spots ranged between 250 for CCB and 800 for SR in the sequence SR > DP ≈ SN > C16-F > CCB. These differences were shown to rely mainly on the sensitivity between dyes leading to the detection of additional spots belonging to classes of lower abundance. Analysis of the distribution of variation coefficients for spots from replicates showed differences in the staining reproducibility between dyes that ranged in the order SR > C16-F > DP > SN > CCB. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.

Frequency-mixing scheme for the production of tunable narrowband XUV radiation (91–95 nm): application to precision spectroscopy and predissociation in diatomic molecules

Tunable narrowband extreme ultraviolet radiation in the range 91–95 nm is produced by sum-frequency mixing of the outputs of a visible pulsed dye amplifier (seeded by a ring dye laser) and of a seeded second-harmonic Nd:YAG laser and subsequent frequency tripling in a gas jet of xenon. The capability of this scheme to provide tunable narrowband extreme ultraviolet radiation is demonstrated in several spectroscopic studies. The bandwidth of this system (0.01 cm-1) is deduced from a recording of absorption spectra of the 4p5(2P1/2) 6d, J=1 line in krypton. The applicability of the system for gas-phase molecular spectroscopic studies is demonstrated in recordings of the Werner bands (4,0) in H2 and (5,0) in D2 at unprecedented absolute accuracy. Line-broadening studies are performed on the b′1Σu+,v=5 valence state in N2, yielding a lifetime of 210±25 ps. A singlet–triplet perturbation, giving rise to an accidental predissociation in an excited 1Π Rydberg state in carbon monoxide at an excitation energy of 107680 cm-1, is analyzed in high resolution.

Fast visible dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels compatible with matrix assisted laser desorption/ionization-mass spectrometry.

A fast and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1- and 2-D SDS-PAGE) is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon (ZC) and ethyl violet (EV) to form an ion-pair complex. The protocol, including fixing, staining and quick washing steps, can be completed in 1-1.5 h depending upon gel thickness. It has a sensitivity of 4-8 ng, comparable to that of colloidal Coomassie Brilliant Blue G (CBBG) staining with phosphoric acid in the staining solution. The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from MS. Considering the speed, sensitivity and compatibility with MS, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

Multidye Nanostructured Material for Optical Data Storage and Security Labeling

We report the design, synthesis, and application of a multidye polymer nanocomposite with respect to high-density 3D optical data storage and security labeling. Core−shell latex particles containing visible and near-IR dyes in the core-forming polymer and the shell-forming polymer were used as the functional building blocks to produce a multicolored multiphase polymeric material with a minimized energy transfer between the dyes. The core−shell particles were prepared by copolymerizing dye-labeled monomers with the hosting polymer. Data recording was achieved using confocal fluorescent microscopy by selective photobleaching of the dyes periodically distributed in the nanocomposite film.

Designing Polymers for Biphasic Liquid/Liquid Separations after Homogeneous Reactions

The phase-selective solubility properties of polymer supports that could be used in thermomorphic and latent biphasic systems useful in synthesis and catalysis were evaluated using polymers tagged with either visible dyes or fluorescent probes. Heptane/DMF, heptane/90% ethanol−water, heptane/ethyl acetate, heptane/ethanol, and heptane/tert-butyl alcohol solvent mixtures were all studied as examples of thermomorphic or latent biphasic systems. A range of polymers including polyisobutylene (PIB), poly(tert-butylstyrene) (PTBS), poly(octadecyl acrylate) (PODA), and poly(octadecyl methacrylate) (PODMA) were tested for hydrophobic phase-selective solubility. The results of these studies are compared to prior work with polar and nonpolar poly(N-alkylacrylamide)s and polystyrene. Together with this prior work, these results show that a wide range of polymers and solvent mixtures can be used for the recycling of soluble polymer-bound catalysts, reagents, and sequestrants using either thermomorphic or latent bipha...

Soil structural interfaces in some Texas Vertisols and their impact on solute transport

Structural surfaces and slickensides have been reported to be important features impacting sorption–desorption of chemicals and bypass flow of pollutants to shallow groundwater aquifers. This study provides visible, microscopic and submicroscopic characteristics of slickenside features in two Texas Vertisols of different geomorphological age. Transport of visible anionic dye and CaBr is studied as related to soil structural surfaces, root channels and slickenside planes. Macromorphic observations were coupled with thin section microscopy, scanning electron microscopy and electron microprobe analyses. Slickenside surfaces differed from subjacent micromass of the ped interiors by stronger preferred orientation of clay particles and reduction in macropores with corresponding increase in micropores. Such characteristics could make slickensides act as semi-permeable barriers to solute transport, but this phenomenon could not be confirmed from CaBr studies herein. The majority of the solute bypass flow in both soils was conducted through root channels and ped interfaces. Slickenside surfaces in these Vertisols conducted less than 1% of the flow. It was observed primarily when the dye solution was delivered to the surface of the slickenside via the adjacent root channel. In spite of the preferential orientation of clay particles along slickenside planes, the penetration of Br into the aggregate interior was not retarded like the larger organic anion dye. These results may reflect specific moisture conditions, which should be considered in future works.