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This study aims to evaluate the clinical value of PCR-fluorescent probes for detecting the mutation gene associated with extensively drug-resistant tuberculosis (XDR-TB). The molecular species identification of 900 sputum specimens was performed using polymerase chain reaction (PCR)-fluorescent probe. The mutations of the drug resistance genes rpoB, katG, inhA, embB, rpsL, rrs, and gyrA were detected. The conventional drug susceptibility testing (DST) and PCR-directed sequencing (PCR-DS) were carried out as control. DST demonstrated that there were 501 strains of rifampicin resistance, 451 strains of isoniazid resistance, 293 strains of quinolone resistance, 425 strains of streptomycin resistance, 235 strains of ethambutol resistance, and 204 strains of amikacin resistance. Furthermore, 427 (47.44%) or 146 (16.22%) strains were MDR-TB or XDR-TB, respectively. The mutations of the rpoB, katG, inhA, embB, rpsL, rrs, and gyrA genes were detected in 751 of 900 TB patients by PCR-fluorescent probe method, and the rate of drug resistance was 751/900 (83.44%). No mutant genes were detected in the other 149 patients. Compared with DST, the mutant rates of rpoB, katG/inhA, rpsL, rrs, embB, and gyrA of six drugs were higher than 88%; five of six drugs were higher than 90% except for SM (88.11%). The MDR and XDR mutant gene types were found in 398 (42.22%) and 137 (15.22%) samples. PCR-DS was also employed and confirmed the PCR-fluorescent probe method with the accordance rate of 100%. The PCR-fluorescent probe method is rapid and straightforward in detecting XDR-TB genotypes and is worthy of being applied in hospitals., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Luminescent derivatives of benzanthrone are becoming more useful based on their light-absorbing and fluorescent-emitting properties. Our previous studies showed that luminescent staining properties of the same benzanthrone dye differ for variable parasite samples. Therefore, two types of benzanthrone dyes were prepared. One has a strongly basic amidine group and a halogen atom, and the other has an amide moiety and a tertiary amine group. Trematoda Parafasciolopsis fasciolaemorpha is a liver fluke of a moose ( Alces alces ) and has a significant influence on the health and abundance of the moose population. Staining protocols for parasite P. fasciolaemorpha specific organ or organ systems imaging are mostly time-consuming and labor-intensive. The study aimed to compare the fixation technique and the staining protocol by synthesized benzanthrone luminescent dyes to determine detailed morphology, anatomical arrangement of the organ systems and gross organization of the muscle layers of P. fasciolaemorpha using confocal laser scanning microscopy. Luminophores were tested for samples fixed in different fixatives. Developed dyes and staining protocol resulting in imaging of all parts of trematode without additional sample preparation procedures, which usually are required for parasite examination. Obtained results confirmed that the most qualitative results could be reached using 3-N-(2-piperidinylacetamido)benzanthrone dye which has amide moiety and a tertiary amine group. Based on obtained results, 3-N-(2-piperidinylacetamido)benzanthrone gave more qualitative parasite visualization than 2-bromo-3-N-(N',N'-dimethylformamidino)benzanthrone.

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom ® FXIII Activity, a functional ammonia release assay, and the HemosIL ™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Two red-emitting dicyanomethylene-4H-pyran (DM) based fluorescent probes were designed and used for peroxynitrite (ONOO - ) detection. Nevertheless, the aggregation-caused quenching effect diminished the fluorescence and restricted their further applications. To overcome this problem, tetraphenylethylene (TPE) based glycoclusters were used to self-assemble with these DM probes to obtain supramolecular water-soluble glyco-dots. This self-assembly strategy enhanced the fluorescence intensity, leading to an enhanced selectivity and activity of the resulting glyco-dot comparing to DM probes alone in PBS buffer. The glyco-dots also exhibited better results during fluorescence sensing of intracellular ONOO - than the probes alone, thereby offering scope for the development of other similar supramolecular glyco-systems for chemical biological studies., (© 2020 Wiley-VCH GmbH.)

Nitric oxide (NO) is an important gaseous signaling molecule related to various human diseases. To investigate the biological functions of NO, many strategies have been developed for real-time monitoring the NO levels in biological systems. Among these strategies, fluorescent probes are considered to be one of the most efficient and applicable methods owing to their excellent sensitivity and selectivity, high spatiotemporal resolution, noninvasiveness, and experimental convenience. Therefore, great efforts have been paid to the design, synthesis, and fluorescence investigation of novel NO fluorescent probes in the past several years. However, few of them exhibit practical applications owing to the low concentration, short half-life, and rapid diffusion characteristics of NO in biological systems. Rational design of NO fluorescent probes with excellent selectivity and sensitivity, low cytotoxicity, long-lived fluorescent emission, and low background interference is still a challenge for scientists all over the word. To provide spatial-temporal information, this article focuses on the progress made in the organic and metal complex-based NO fluorescent probes during the past five years. The key structural elements and sensing mechanisms of NO fluorescent probes are discussed. Some novel ratiometric, luminescence, and photoacoustic probes with low background interference and deep tissue penetrating ability are mentioned. All these probes have been used for imaging exogenous and endogenous NO in cells and animal models. More importantly, this article also describes the development of multi-functional NO fluorescent probes, such as organelle targeting probes, dual-analysis probes, and probe-drug conjugates, which will inspire the design of various functional fluorescent probes.

The fluorescence detected using fluorochrome-labelled monoclonal antibodies depends not only on the abundance of the target antigen, but amongst many other factors also on the effective fluorochrome-to-antibody ratio. The diagnostic approach of the EuroFlow consortium relies on reproducible fluorescence intensities over time. A capture bead system for mouse immunoglobulin light chains was utilized to compare the mean fluorescence intensity of 1323 consecutive antibody lots to the currently used lot of the same monoclonal antibody. In total, 157 different monoclonal antibodies were assessed over seven years. Median relative difference between consecutive lots was 3.8% (range: 0.01% to 164.7%, interquartile range: 1.3% to 10.1%). The relative difference exceeded 20% in 8.8% of all comparisons. FITC labelled monoclonal antibodies (median relative difference: 2.1%) showed a significantly smaller variation between lots than antibodies conjugated to PE (3.5%), PECy7 (3.9%), PerCPCy5.5 (5.8%), APC (5.8%), APCH7 (7.4%), and APCC750 (14.5%). Reagents labelled with Pacific Blue (1.4%), Pacific Orange (2.4%), HV450 (0.7%), and HV500 (1.7%) demonstrated more consistent results compared to conjugates of BV421 (4.1%) and BV510 (16.2%). Additionally, significant differences in lot-to-lot fluorescence stability amongst antibodies labelled with the same fluorochrome were observed between manufacturers. These observations might guide future quality recommendations for the production and application of fluorescence-labelled monoclonal antibodies in multicolor flow cytometry., (Copyright © 2017. Published by Elsevier B.V.)

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Near-infrared (NIR) responsive agents for cancer bioimaging and photothermal therapy are available and significant. Herein, we employed two easily available dyes, boron-dipyrromethane and coumarin, to synthesize a pair of coumarin-borondipyrromethane dyes with different conjugate degrees (BDC and BSC). The difference in conjugate degree made their photophysical properties poles apart. After the self-assembling of BDC and BSC, the newly constructed nanoparticles (BDC NPs and BSC NPs) demonstrated good biocompatibility. Moreover, BDC NPs exhibited a good photothermal effect under irradiation of 808 nm laser, which could effectively inhibit the growth of HeLa cells, and BSC NPs could quickly show a conspicuous fluorescence in the HeLa cells. The exploration demonstrates that the two organic dyes prepared with different conjugation degrees could provide new options for photothermal therapy of cancer and rapid bioimaging.

A novel near-infrared (NIR) ratiometric fluorescent probe HBT-GSH derived from conjugated benzothiazole was developed for the selective detection of glutathione (GSH) over cysteine (Cys) and homocysteine (Hcy). The probe was sophisticatedly designed based on the GSH selectively induced enhancement of intramolecular charge transfer (ICT) fluorescence. It was synthesized by masking the active phenol group of 2,6-bis(2-vinylbenzothiazolyl)-4-fluorophenol through an acetyl group that acts both as a trigger of the ICT fluorescence and as a recognition moiety for GSH. On its own, the probe HBT-GSH exhibited strong blue fluorescence emission at 426 nm and weak NIR fluorescence emission at 665 nm in aqueous solution, whereas the NIR fluorescence was significantly enhanced and the short emission decreased upon the addition of GSH. Thus an NIR ratiometric fluorescent probe for GSH was developed based on the GSH-selective removal of the acetyl group, therefore switching on the ICT in HBT-GSH. The fluorescence intensity ratio (I 665 nm /I 426 nm ) showed a linear relationship with a GSH concentration of 0-100 μM with a detection limit of 0.35 μM. Moreover, the fluorescent probe was successfully used for the ratiometric fluorescence bioimaging of GSH in living cells.

Yellow-emissive carbon dots (Y-CDs) were prepared by a solvothermal method using anhydrous citric acid and 2,3-phenazinediamine as the starting materials. The Y-CDs display a 24% fluorescence quantum yield, a 188-nm Stokes' shift and excellent stability. They are shown here to be excellent fluorescent probes for the determination of Ag(I) ion and glutathione (GSH). If exposed to Ag(I) ions, they are bound by the carboxy groups of the Y-CDs, and this causes quenching of fluorescence (with excitation/emission maxima at 380/568 nm) via a static quenching mechanism. This effect was used to design a fluorometric assay for Ag(I). The quenched fluorescence of the Y-CDs can be restored by adding GSH due to the high affinity of GSH for Ag(I). The calibration plot for Ag(I) is linear in the 1-4 μM Ag(I) concentration range, and the limit of detection is 31 nM. The respective values for GSH are 5-32 μM, and 76 nM, respectively. The method was applied to the detection of Ag(I) in spiked environmental water samples and gave recoveries ranging from 93 to 107%. It was also applied to the determination of GSH in tomatoes and purple grapes and gave satisfactory recoveries. The Y-CDs display low cytotoxicity and were successfully used to image Ag(I) and GSH in H1299 cells. Graphical abstract Schematic presentation of the mechanism of yellow fluorescent CDs for the detection of Ag + and glutathione.

The authors describe a fluorometric method for the turn-on determination of vitamin C (ascorbic acid). The blue fluorescence of silicon nanoparticles (SiNPs; with excitation/emission maxima at 350/450 nm) is found to be quenched by CoOOH nanoparticles (NPs). In the presence of vitamin C, the CoOOH NPs are decomposed by a redox reaction between the diol group of vitamin C and CoOOH NPs. As a result, fluorescence recovers. On the basis of this finding, a fluorometric method was designed for the turn-on detection of vitamin C. Under optimal conditions, the method has a low detection limit (0.47 μM) and a linear response in the 0.5 μM to 20 μM a concentration range. It was successfully applied to the determination of vitamin C in spiked red grape and orange juice, and in vitamin C tablets. Graphical abstract A target-triggered dissociation of quencher-based strategy for the fluorescence "turn-on" detection of vitamin C was developed. It is based on surface energy transfer (SET) and an inner filter effect (IFE) between silicon nanoparticles and CoOOH nanoparticles as well as the redox reaction between vitamin C and CoOOH nanoparticles.

A highly sensitive method for brief and economical staining of proteins in 1-D and 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by 4',5'-Dibromofluorescein (DBF) was developed in this study. Down to 0.025-0.05ng protein could be detected within 10min (only 2 steps) by DBF stain, which is approximately 10-fold more sensitive than those of Eosin Y (EY) and SYPRO Ruby stains, and 20-fold more sensitive than that of imidazole-zinc (IZ) negative stain. In addition, the LC-MS/MS results indicated that the newly developed staining method is compatible with the downstream protein identification. Moreover, the mechanism of DBF stain was investigated by molecular docking., (Copyright © 2017 Elsevier B.V. All rights reserved.)

CE equipment detects and deconvolutes mixtures containing up to six fluorescently labeled DNA fragments. This deconvolution is done by the collection software that requires a spectral calibration file. The calibration file is used to adjust for the overlap that occurs between the emission spectra of fluorescence dyes. All commercial genotyping and sequencing kits require the installation of a corresponding matrix standard to generate a calibration file. Due to the differences in emission spectrum overlap between fluorescent dyes, the application of existing commercial matrix standards to the electrophoretic separation of DNA labeled with other fluorescent dyes can yield undesirable results. Currently, the number of fluorescent dyes available for oligonucleotide labeling surpasses the availability of commercial matrix standards. Therefore, in this study we developed and evaluated a customized matrix standard using ATTO 633, ATTO 565, ATTO 550, ATTO Rho6G, and 6-FAM dyes for which no commercial matrix standard is available. We highlighted the potential genotyping errors of using an incorrect matrix standard by evaluating the relative performance of our custom dye set using six matrix standards. The specific performance of two genotyping kits (UniQTyper™ Y-10 version 1.0 and PowerPlex® Y23 System) was also evaluated using their specific matrix standards. The procedure we followed for the construction of our custom dye matrix standard can be extended to other fluorescent dyes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Plasmin is a protease in milk, which concentration is increased during mastitis. The aim of the study was design and characterize short, tailor made fluorogenic substrates for plasmin to see differences in plasmin activity in healthy milk compared to mastitic milk. According to the specificity matrix of plasmin, containing 125 currently known plasmin substrates, six novel plasmin specific substrates were designed and characterized. Eight other substrates were insensitive for plasmin, which was in line with our expectations. The plasmin-sensitive substrates showed no cross-reactivity with proteases from mastitogenic bacteria in vitro. A proof of concept to distinguish healthy milk from mastitic milk is described. Potentially, these substrates present a rapid alternative to the currently established methodologies to detect mastitis in milk.

Fluorescent Ca(2+) indicators are widely used to measure the concentration of free Ca(2+) ([Ca(2+)]free) in biological processes. By calibrating the dye under the same experimental conditions as employed during its planned use, the actual [Ca(2+)] can be calculated from the measured fluorescence. When using non ratiometric dyes, such as the Oregon Green BAPTA (OGB) family of dyes or the Fluo dyes, the steady-state affinity (K(d)) and the ratio between the maximal and minimal fluorescence (F(ratio) = F(max)/F(min)) of the particular dye are needed for this conversion. Although these values are usually given by the manufacturer, we consistently find that the actual values can differ between various batches delivered by the companies that make the dyes. In this protocol, we provide the recipe for a series of solutions with a known and tightly buffered [Ca(2+)](free) and describe how to use these mixtures to determine the exact K(d) and F(ratio) of a fluorescent Ca(2+) dye., (© 2014 Cold Spring Harbor Laboratory Press.)

Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo., (© 2014 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: The stability and performance of tandem-conjugated antibodies can be impaired when stored in antisera cocktails (Biancotto et al., J Immunol Methods 2011;363:245-261; Rawstron et al., Leukemia 2013;27:142-149). This, and the need for frequent re-compensation due to the possible spectral spillover variation between tandem lots, reduces the robustness of clinical flow cytometry panels that include tandems. Since tandems are required for standard 8-10 color screens, further studies of the stability of tandems in cocktails and their spillover variability are warranted., Methods: The performance of PE- and APC-tandems stored in cocktails was tested on fresh bone marrow, preserved blood and lyophilized cell samples over 1-, 6-, or 8-week periods, respectively, and their spillover matrices were compared. The observed correction factor differences were used as the basis for analyzing how the application of an incorrect compensation matrix could influence data interpretation., Results: Signal intensities and background fluorescence remained constant for all fluorochromes in the cocktails tested. Spillover correction factors for different PE-Cy7 mAbs did not exceed or were only marginally higher than those for non-tandem organic dye-conjugated mAb. By applying the correction factor differences observed between tandem mAb lots to clinical data, it was found that the over and under compensation would not alter the clinical interpretation., Conclusions: Tandems can be safely stored and used in cocktails. However, each cocktail should be tested on relevant material prior to use. Exact compensation settings are a requirement for accurate data. Provided that careful evaluation of tandem compensation requirements is carried out, certain tandems may use a generic compensation matrix., (© 2013 Clinical Cytometry Society.)

This paper describes a simple and efficient approach to reduce the background level of confocal laser induced fluorescence (LIF) detection for round capillaries by laterally shifting the laser focus point. A phenomenon of spontaneous separation of the fluorescence and reflected laser beams at the pinhole of a confocal LIF system when the laser focus point deviates from the center of a capillary channel to the sides was observed for the first time. On the basis of this phenomenon, the reflected laser light from the capillary-air interfaces could be mostly eliminated with a spatial filtering pinhole. A comprehensive study on the phenomenon and optimization of the shift distance was carried out using both experimental and simulation methods. A best shift distance of ±20 μm was obtained, with which background intensity could be significantly reduced by 98.9%, while fluorescence intensity was only reduced by 25.7%, resulting in an improvement of signal-to-noise ratio of 8.3 times, compared with that at a shift distance of 0 μm usually used in most of the confocal LIF systems for round capillaries. A limit of detection of 66 fM was obtained for sodium fluorescein. To demonstrate its potential as an on-column sensitive detector for microscale separation systems, the present system was coupled with a capillary electrophoresis system for separation of four fluorescein isothiocyanate labeled amino acids with concentrations of 100 pM.

Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb(3+)). Single-stranded oligonucleotides greatly enhance the Tb(3+) emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb(3+)/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb(3+), producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb(3+)/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb(3+)/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36±1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes. In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.

We studied the collisional quenching of the erythrosine B fluorophore by potassium iodide. The quenching follows a Stern-Volmer dependence up to the highest quencher concentration. The lifetime of erythrosine B decreases to 24 ps in 5.02 M of potassium iodide. The quantum yield of erythrosine B in the presence of 5.02 M KI is 0.0035. The relatively high brightness makes this compound attractive as an ultrashort reference in time-resolved measurements. In both frequency- and time-domain fluorescence techniques, there is a need for lifetime standards with extremely short decay times. Mimicking the instantaneous scattering at longer wavelengths allows color-effect-free measurements in the emission region. Another motivation is the problem of obtaining the impulse response function in the case of two-photon excitation. Time-resolved microscopy also benefits from fast-decaying dyes because the impulse response function can be evaluated at the emission wavelength of the investigated specimen without changing filters. We demonstrated that impulse response functions for commonly used detectors are practically the same for scattering as for quenched erythrosine B emission. We also analyzed a complex fluorescence decay using both elastic scattering and quenched erythrosine B emission as a response function.

An extensive study is described to identify the most suitable fluorescent label in magnetic microsphere sedimentation arrays. The investigated fluorescent labels, commonly used in multiplex analysis, include organic dyes, (fluorescein, Alexa488, Cy5) fluorescent proteins (R-Phycoerythrin, Allophycocyanine, PBXL-3) polymer nanoparticles (FluoSpheres, PD-Pt) and semiconductor nanocrystals (Quantum dots). DNA hybridization assays on magnetic microspheres were applied as model systems to reveal label performance. The fluorescent labels were characterized under optimized conditions regarding signal intensity, non-specific binding and photo-stability. The advantages and drawbacks of individual labels are discussed. The limit of detection and dynamic ranges are determined to compare the performance of selected labels. Detection limits of 2 x 10(-10) mol/L are found for the determination of oligonucleotides using PBXl-3 as label, which is comparable with typical flow cytometer systems. The results and protocols are highly valuable for any type of bead based assays and can be easily transferred.

In this study, the association behavior of pyrene with different dissolved humic materials (DHM) was investigated utilizing the recently developed segmented frequency-domain fluorescence lifetime method. The humic materials involved in this study consisted of three commercially available International Humic Substances Society standards (Suwannee River fulvic acid reference, SRFAR, Leonardite humic acid standard, LHAS, and Florida peat humic acid standard, FPHAS), the peat derived Amherst humic acid (AHA), and a chemically bleached Amherst humic acid (BAHA). It was found that the three commercial humic materials displayed three lifetime components, while both Amherst samples displayed only two lifetime components. In addition, it was found that the chemical bleaching procedure preferentially removed red wavelength emitting fluorophores from AHA. In regards to pyrene association with the DHM, different behavior was found for all commercially available humics, while AHA and BAHA, which displayed strikingly similar behavior in terms of fluorescence lifetimes. It was also found that there was an enhancement of pyrene's measured lifetime (combined with a decrease in pyrene emission) in the presence of FPHAS. The implications of this long lifetime are discussed in terms of (1) quenching mechanism and (2) use of the fluorescence quenching method used to determine the binding of compounds to DHM.

We present absolute two-photon absorption (2PA) spectra of 15 commercial organic dyes covering an extended range of excitation wavelengths, 550-1600 nm. The 2PA is measured with an estimated accuracy +/-10% using a femtosecond fluorescence excitation method. The data are corrected for the variations of the pulse duration and the beam profile with the excitation wavelength, and are applicable as reference standards for 2PA measurements.

DNA-directed chemical ligations provide the opportunity to diagnose DNA sequences with very high sequence specificity. Fluorescent labels have been attached to reactive probes to enable the homogeneous detection of DNA and RNA. However, it has frequently been found that the attachment of fluorescent labels results in decreases of ligation fidelity. Herein we describe the development of a fluorogenic ligation reaction that provides for 10(2)-fold to perfect sequence selectivity. The reaction is based on the isocysteine-mediated native chemical PNA ligation. It is shown that DNA-induced rate accelerations of approximately 43.000-fold can be obtained through subtle variations of the ligation conditions. PNA-thioesters and isocysteine-PNA conjugates were labeled with FAM and TMR fluorophores, respectively. For gaining rapid synthetic access, a convenient on-resin labeling approach was developed. A new PNA monomer featuring an Alloc-protected lysine side chain was synthesized and coupled in solid-phase PNA synthesis. In the event of a ligation reaction the two fluorophores are brought into proximity. It is shown that fluorescence resonance energy transfer provides a positive fluorescence signal which is specific for product formation rather than for loss of starting materials. Single base mutations can be detected within minutes and with very high sequence selectivity at optimized conditions.

Optimizing the amount of primer to use in PCR amplification is one of the most important steps when developing protocols for genetic profiling, where subtle changes in primer concentration result in major impacts on the amount of desired product that is amplified. However; there are frequently discrepancies between the reported and actual quantity of primers delivered by suppliers, resulting in a need for re-optimization of conditions between primer orders and limiting the ability to standardize conditions between laboratories. To increase the consistency of genetic profiling protocols, we have developed a simple method to assess the quantity and quality of fluorescently labeled primers and therefore standardize reaction conditions through time and across laboratories. The method is based on analysis by electrophoresis with an automated fluorescent DNA analyzer.

Although synaptically released, vesicular Zn(2+) has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn(2+) release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn(2+) at the mossy fiber (MF) synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn(2+) indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green [K(DZn2+) approximatelly 1 microM] , Zinpyr-4 K(DZn2+) approximately 1 nM and FluoZin-3 K(DZn2+) approximately 15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca(2+) K(DCa2+) approximately 200-300 microM which was present in the extracellular medium ([Ca(2+)](o)>2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically released Zn(2+) in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn(2+) onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn(2+) contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system.

Proteomic analyses using two-dimensional gel electrophoresis (2DE) depend heavily upon the quality of protein stains for sensitive detection. Indeed, detection rather than protein resolution is likely a current limiting factor in 2DE. The recent development of fluorescent protein stains has dramatically improved the sensitivity of in-gel protein detection and has enabled more accurate protein quantification. Here, we have evaluated the overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo, and the most commonly used stain, Sypro Ruby (SR). All stains were found to be statistically comparable with regard to number of protein spots detected, but SR was superior with regard to fluorophore stability (e.g., capacity for repeated use of the stain solution). Notably, colloidal Coomassie Blue was also found to be comparable to SR when detected using an infrared fluorescence imaging system rather than standard densitometry. Thus, depending on available equipment and operating budgets, there are at least two high-sensitivity alternatives to achieve the best currently available in-gel protein detection: Sypro Ruby or Coomassie Blue.

With the Calibration Kit Spectral Fluorescence Standards BAM-F001-BAM-F005, we developed a simple tool for the characterization of the relative spectral responsivity and the long-term stability of the emission channel of fluorescence instruments under routine measurement conditions thereby providing the basis for an improved comparability of fluorescence measurements and eventually standardization. This first set of traceable fluorescence standards, which links fluorescence measurements to the spectral radiance scale in the spectral range of 300-770 nm and has been optimized for spectrofluorometers, can be employed for different measurement geometries and can be adapted to different fluorescence techniques with proper consideration of the underlying measurement principles.

In the context of clinical trials, calibration protocols for optical instruments that ensure measurement accuracy and the ability to carry out meaningful comparisons of data acquired from multiple instruments are required. A series of calibration standards and procedures are presented to assess technical feasibility of optical devices for cervical precancer detection. Measurements of positive and negative standards, and tissue are made with two generations of research grade spectrometers. Calibration accuracy, ability of standards to correct and account for changes in experimental conditions, and device components are analyzed. The relative frequency of measured calibration standards is investigated retrospectively using statistical analysis of trends in instrument performance. Fluorescence measurements of standards and tissue made with completely different spectrometers show good agreement in intensity and lineshape. Frequency of wavelength calibration standards is increased to every 2 h to compensate for thermal drifts in grating mount. Variations in illumination energy detected between standards and patient measurements require probe redesign to allow for simultaneous acquisition of illumination power with every patient measurement. The use of frequent and well-characterized standards enables meaningful comparison of data from multiple devices and unambiguous interpretation of experiments among the biomedical optics community.

Nonspecific binding is a frequently encountered problem with fluorescent labeling of tissue cultures when labeled with quantum dots. In these studies various cell lines were examined for nonspecific binding. Evidence suggests that nonspecific binding is related to cell type and may be significantly reduced by functionalizing quantum dots with poly(ethylene glycol) ligands (PEG). The length of PEG required to give a significant reduction in nonspecific binding may be as short as 12-14 ethylene glycol units.

Expression microarrays have great potential for clinical use but variability of the results represents a challenge for reliable practical application. The amount of fluorescent dye used in microarray experiments is a significant source of variability that has not been systematically studied. Here we demonstrate that the quantity of Cy3 dye affects microarray results performed on tumor specimens. Signal-to-noise ratios and coefficients of variation are significantly improved by increasing Cy3 to 150-180 pmol, but any further increase does not improve the data. In conclusion, optimal amounts of dye reduce variability and improve reliability of expression microarray experiments.

The basis of the selectivity of fluorochromes routinely used to visualize the endoplasmic reticulum (ER) in live cells remains obscure. To clarify this, interactions of living cells with fluorochromes of varied physicochemical properties were analyzed experimentally and numerically using a quantitative structure activity relationship analysis (QSAR). Routine selective ER probes were found to be amphipathic, lipophilic cations with moderate-sized conjugated systems. The moderately lipophilic character permits probe uptake by passive diffusion without nonspecific accumulation in biomembranes. The moderately amphipathic character favors uptake into the ER, perhaps owing to its high concentration of zwitterionic lipid head-groups. The QSAR model rationalizes the impractical character of some ER probes mentioned in the literature, and could permit design of novel ER probes with different emission colors. The possibility of using the QSAR model as a tool to predict the accumulation of xenobiotics in the ER of living cells is illustrated by the localization of certain antipsychotic drugs in cultured cells.

Dye switching and biological replication substantially increase the cost and the complexity of cDNA microarray studies. The objective of the present analysis was to quantitatively assess the importance of these procedures to provide a quantitative basis for decision-making in the design of microarray experiments. Taking advantage of the unique characteristics of a published data set, the impact of these procedures on the reliability of microarray results was calculated. Adding a second microarray with dye switching substantially increased the correlation coefficient between observed and predicted ln(ratio) values from 0.38 +/- 0.06 to 0.62 +/- 0.04 (n = 12) and the outlier concordance from 21 +/- 3% to 43 +/- 4%. It also increased the correlation with the entire set of microarrays from 0.60 +/- 0.04 to 0.79 +/- 0.04 and the outlier concordance from 31 +/- 6% to 58 +/- 5% and tended to improve the correlation with Northern blot results. Adding a second microarray to include biological replication also improved the performance of these indices but often to a lesser degree. Inclusion of both procedures in the second microarray substantially improved the consistency with the entire set of microarrays but had minimal effect on the consistency with predicted results. Analysis of another data set generated using a different cDNA labeling method also supported a significant impact of dye switching. In conclusion, both dye switching and biological replication substantially increased the reliability of microarray results, with dye switching likely having even greater benefits. Recommendations regarding the use of these procedures were proposed.

Fluorescence labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide was shown to be a promising approach toward the accurate determination of carbonyls in cellulosic materials. Combined with gel permeation chromatography in DMAc/LiCl with fluorescence/multiple-angle laser light scattering/refractive index detection, the method yields carbonyl profiles relative to the molecular weight of the cellulosic material. The derivatization procedure can be carried out either homogeneously in DMAc/LiCl or advantageously as heterogeneous derivatization in aqueous buffer. The heterogeneous carbonyl group determination, offering shorter reaction times and increased simplicity as compared to the homogeneous approach, was comprehensively validated. The carbonyl content in numerous dissolving pulps of different provenience has been determined, including pulps with carbonyl contents additionally increased by oxidative treatment. The method was also applied to follow bleaching sequences and oxidative treatments of pulps.

The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.

Fluorescent pteridines are photosensitive pigments of mosquito cuticle. Their quantity decreases with time during the adult life of mosquitoes. In order to test the feasibility of the reversed-phase HPLC (High Pressure Liquid Chromatography) technique, to measure pteridine fluorescence and to estimate the calendar age, reared Anopheles gambiae of known age are used. Head and thorax were studied on mosquitoes aged 0, 5, 10, 20 days after emergence. There was significant difference between male and female of 0 and 5 days of age; the difference was not significant at 10 days of age. The level of fluorescence between a female's head and thorax non separated does not differ from the sum of fluorescence level of separated head and thorax. Pteridin fluorescent of female's head and thorax does not correlate. It decreases significantly with chronological age between 0 and 5 days either for head + thorax or for head alone. Conversely, this fluorescence quantity is weak and maintained constant beyond 5 days. The use of thorax alone of the female mosquito allows the differentiation of mosquito aged less than 5 days, between 5 and 10 days and aged more than 10 days. Reversed-phase HPLC technique, at least in the way we have demonstrated, does not appear sensitive enough to estimate the age of the species An. gambiae reared in an insectarium. It seems that this technique, relatively complex to manage, does not bring a substantial advantage compared to the method of Detinova, which allows the separation of nulliparous and parous females (i.e. < or = 3 days versus > 3 days).

To evaluate the origin of cells after allogeneic haematopoietic stem cell transplantation we optimised and evaluated two commercially available systems (AmpFlSTR Profiler Plus and GenePrint Powerplex-16) which are based on multiplex fluorescent short tandem repeat (STR) analysis. A standard procedure for interpretation of electropherographs was found essential to obtain reproducible results. On the basis of the relative length of donor and recipient alleles, TYPE-I (no shared alleles are used to calculate chimerism), TYPE-II (one shared and one unshared allele is used to calculate chimerism) or TYPE-III (not informative) allelic distribution types were distinguished. Also, stutter peaks were recognised as an important criterion to exclude a marker for analysis. Intralaboratory and multicentre evaluation of the AmpFlSTR Profiler Plus system showed that mixed blood samples could be determined with an absolute deviation of less than 2%. A sensitivity threshold was set at 5% for TYPE-I and 10% for TYPE-II markers since relative imprecision increases at low chimerism values. No significant difference of calculated chimerism values was observed between STR markers shared between both systems. By monitoring 26 allogeneic peripheral blood stem cell transplants, the applicability of the proposed method was demonstrated.

The viability of using thiazole orange as an alternative to ethidium bromide in a fluorescent intercalator displacement (FID) assay is explored by profiling the DNA binding affinity and sequence selectivity of netropsin. Utilizing a library of hairpin deoxyoligonucleotides containing all possible four base-pair sequences, the method provides a high resolution profile of the DNA binding properties of small molecules in a high throughput format.

Fluorescence intensity of indocyanine green (ICG) derivative (ICG-sulfo-OSu) was too low for its use to detect microlesions. Therefore, we examined the effects of reinforcement agents on ICG-sulfo-OSu labeled antibodies. Solutions of distearoylphosphatic acid sodium salt (DSPA) and octylglucoside (OG) in physiological phosphate buffered saline (PBS) were found to increase the intensity of fluorescence of ICG-sulfo-OSu labeled antibodies, with shift in the fluorescence peak wavelength from 804 to 821 nm.

The Biological Stain Commission occasionally has been requested to certify fluorochromes as biological stains. Although formal certification is unlikely in the near future, the Commission is nevertheless concerned with the quality of these reagents. Commercial samples of fourteen fluorochromes were investigated for the presence of fluorescent organic impurities using reverse phase thin layer chromatography. Our findings suggest that some fluorochrome dyes are pure, but most are impure. Most fluorochromes vary in purity among vendors and among batches sold by single vendors. Impurities may be present at such high concentration that little of the presumed compound is present. Some impurities behave quite differently from the nominal dye. This may either create confusion or it might be useful. In the latter case, however, the impurity may occur only in a single batch. Impurities result from problems related to organic syntheses, separations, and economics. Solving those problems is often expensive, and what is expensive may not be performed. Fortunately, knowledge of synthetic chemistry often permits identification of fluorochromes likely to be impure. Moreover, predictions of likely staining effects of particular impurities can be made if appropriate structure-activity models are available. Possible actions by the Commission aimed at limiting the problems resulting from impurities of fluorescent dyes are noted.