How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards.     

Comparison of Two Tricarbocyanine-Based Dyes for Fluorescence Optical Imaging

Optical technologies are evolving in many biomedical areas including the biomedical imaging disciplines. Regarding the absorption properties of physiological molecules in living tissue, the optical window ranging from 700 to 900 nm allows to use fluorescent dyes for novel diagnostic solutions. Here we investigate the potential of two different carbocyanine-based dyes fluorescent in the near infrared as contrast agents for in vivo imaging of subcutaneously grown tumours in laboratory animals. The primary aim was to modify the physicochemical properties of the previously synthesized dye SIDAG to investigate the effect on the in vivo imaging properties.     

Current Problems and Potential Techniques in In Vivo Glucose Monitoring

Accurate in vivo monitoring of glucose concentration would be a valuable asset, particularly for management of diabetes and preterm infants during critical care. In vivo glucose monitoring devices can be divided into two categories: implanted and non-invasive. Extensive research into in vivo glucose monitoring over recent decades has not resulted in the widespread use of clinically reliable monitoring systems. For implanted devices, poor biocompatibility of the materials used for fabrication remains a major challenge, whilst progress in the commercial development of non-invasive devices is hampered by the problem of multiple interference between the detected signals and the biological components. In this review, the methods available for in in-vivo glucose monitoring are described and the associated problems are discussed.     

Development of a Resonator with Automatic Tuning and Coupling Capability to Minimize Sample Motion Noise for in Vivo EPR Spectroscopy

EPR spectroscopy has been applied to measure free radicals in vivo; however, respiratory, cardiac, and other movements of living animals are a major source of noise and spectral distortion. Sample motions result in changes in resonator frequency, Q, and coupling. These instabilities limit the applications that can be performed and the quality of data that can be obtained. Therefore, it is of great importance to develop resonators with automatic tuning and automatic coupling capability. We report the development of automatic tuning and automatic coupling provisions for a 750-MHz transversely oriented electric field reentrant resonator using two electronically tunable high Q hyperabrupt varactor diodes and feedback loops. In both moving phantoms and living mice, these automatic coupling control and automatic tuning control provisions resulted in an 8- to 10-fold increase in signal-to-noise ratio.     

Optical Sensor for the Determination of Glucose Based on KIO4 Chemiluminescence Detection

A method to determine glucose using an optical sensor prepared by entrapping glucose oxidase into silica sol-gel column has been developed. The silica sol-gel film was coated on alumina substrate. The optical sensor is based on the chemiluminescence intensity from the reaction of periodate and hydrogen peroxide in K2CO3 medium. The effect of the ratio of water and alcohol for the preparation of TEOS sol on chemiluminescence intensity was investigated. The effects of pH of enzyme reactor, concentrations of potassium periodate and SDS, and flow rate on the chemiluminescence intensity were studied to find the optimum experimental conditions to determine glucose. The chemiluminescence intensity increased linearly with increasing glucose concentration from 5.0 x 10(-4) M to 1.0 x 10(-7) M and the detection limit was 4.0 x 10(-8) M. Interference effects from some metal ions on chemiluminescence intensity were also investigated.     

Investigation of a Fluorescence Signal Amplification Mechanism Used for the Direct Molecular Detection of Nucleic Acids

A fluorescence signal amplification mechanism allowing detection limits for DNA in the zeptomolar range was investigated. Photophysical properties of the molecular system were studied in order to better explain the signal amplification that is observed. We show that the confinement of a fluorescent DNA hybridization transducer in aggregates improves its quantum yield and photostability. Furthermore, we show that the combination of the resonance energy transfer occurring within the aggregates with the use of a conjugated polymer as the hybridization transducer and donor allows ultrafast and efficient energy coupling to the aggregates and can lead to the excitation of a large number of acceptors by only one donor.     

In Situ Detection of the Pathogen Indicator E. coli Using Active Laser-Induced Fluorescence Imaging and Defined Substrate Conversion

To remotely sense the pathogen indicator E. coli in aquatic systems, we used laser-induced fluorescence imaging to detect the signature produced by the in situ conversion of the defined substrate medium ECMUG. The presence of the enzyme -glucuronidase, indicated by the activity of the fluorogenic 4-methylumbelliferyl (4MU)--D-glucuronide (MUG), is specific to the presence of this organism. Substrate conversion was accomplished in a small stream impacted by non-point source wastewater inputs. Sample chambers slowly inoculated source water with 100 ml of ECMUG media in direct sunlight. Luminescence spectroscopy monitored conversion activity and detected liberation of the 4 MU fluorochrome in 2.5 hours. Detection by laser-induced fluorescence imaging followed at dusk and indicated bright blue emissions typical for converted media. This technique lays the foundation for active remote sensing of source water contamination.     

Fluorescence quenching of 6-methoxyquinoline: an indicator for sensing chloride ion in aqueous media

The quenching of fluorescence intensity and decay time of protonated form of 6-methoxyquinoline (6MQ⁺) with chloride ion (Cl⁻) in aqueous solution at ambient temperatures have been investigated. The quenching follows linear Stern-Volmer relation. The values of Stern-Volmer quenching constant/quenching efficiency (K_sv) and quenching rate constant (K_q) for the Cl⁻ ion are close to 75 M⁻¹ and 3.21×10⁹ M⁻¹ S⁻¹, respectively. The quenching is found to be collisional or dynamical in nature. The study reveals that the system can be used as a sensor for the detection of chloride ion.     

Dictyostelium Extracellular Vesicles Containing Hoechst 33342 Transfer the Dye into the Nuclei of Living Cells: A Fluorescence Study

Cells of the eukaryotic unicellular microorganism Dictyostelium discoideum are constitutively resistant to vital staining of their nuclei by the DNA-specific dye Hoechst 33342. By studying the mechanisms of this resistance, we evidenced that these cells expel vesicles containing the dye for detoxification (Tatischeff et al., Cell Mol Life Sci, 54: 476-87, 1998). The question to be addressed in the present work is the potential use of these extracellular vesicles as a biological drug delivery tool, using Hoechst 33342 as a model of a DNA-targeting drug. After cell growth with or without the dye, vesicles were prepared from the cell-free growth medium by differential centrifugation, giving rise to two types of vesicles. Negative staining electron microscopy showed their large heterogeneity in size. Using fluorescence techniques, data were obtained on the dye loading and its environment inside the vesicles. By UV video-microscopy, it was demonstrated that the dye-containing vesicles were able to deliver it into the nuclei of naive Dictyostelium cells, thus overcoming their constitutive resistance to the free dye. A vesicle-mediated dye-transfer into the nuclei of living human leukaemia multidrug resistant K562r cells was also observed.     

Optimization of the Near-Infrared Fluorescence Labeling for In Vivo Monitoring of a Protein Drug Distribution in Animal Model

The objective of this study is to optimize the parameters in labeling near-infrared (NIR)fluorescent dye cypate to protein drugs for in vivo optical imaging of drug distributions in animal model. l-ASparaginase (l-ASNase) was used as a protein drug model for the study. To achieve this goal, various labeling conditions, including different catalysts, feed ratios of all components, pH conditions, temperatures, and reacting durations, were investigated. The dye-to-protein (D/P) ratio and enzymatic activity were designated as the metric to evaluate the labeling process. The stability of the cypate–protein conjugate in blood serum and its distribution in small animals were subsequently inspected. Results showed that feed ratio of l-ASNase and the pH value played the most important role in adjusting the labeling efficiency. Reaction duration and temperature had less effect on the dye-to-protein labeling properties. The optimal condition for the labeling of cypate to l-ASNase was 4 h reaction duration at 4 °C and pH 8.5 under catalysis by HOBt/HBTU. The dynamic distribution in animal model displayed that the labeled l-ASNase firstly accumulated in liver and cleared from the enteron system. This study demonstrated that the NIR image system combined with NIR probe has the capability to trace the dynamics of protein drugs in animals for drug development.     

聚氨酯/硫化银纳米复合材料的合成及荧光传感性能

通过生物矿化合成了聚氨酯/Ag2S纳米复合薄膜．通过傅里叶变换红外光谱研究、扫描量热法(DSC)、扫描电镜等方法研究了硫化银纳米粒子对复合薄膜物理性质的影响．用DSC 测定了复合材料的热 稳定性．通过对纳米复合材料荧光性能的研究发现薄膜对Ni(II)的存在非常敏感,少量Ni(II)离子的存在使得荧光光谱强度迅速增加．可以预测此复合薄膜可被开发成水溶液中Ni(II)的传感器．     

In Vivo3D LocalizedC Spectroscopy Using Modified INEPT and DEPT

The 3D localized¹³C spectroscopy methods LINEPT and LODEPT, which are modifications of INEPT and DEPT, are proposed. As long as a¹³C inversion pulse (180-degree pulse) is applied at 1/(4J) before the proton echo time in LINEPT and a¹³C excitation pulse (90-degree pulse) is applied at 1/(2J) before the proton echo time in LODEPT, the proton echo time can be set to any value longer than 1/(2J) in LINEPT and longer than 1/Jin LODEPT. As a result, the proton and the¹³C pulses can be applied separately and these proton pulses can be made slice-selective pulses. These localization features of LINEPT and LODEPT were evaluated using a phantom consisting of a cylinder filled with ethanol placed inside another cylinder filled with oil, and localized ethanol spectra could be obtained.In vivo3D localized¹³C spectra from the brain of a monkey could be obtained using decoupled LINEPT, and glutamate C-4 appeared directly after the administration of glucose C-1, followed by the appearance of glutamate C-2, C-3 and glutamine C-2, C-3, C-4.     

<Emphasis Type="Italic">In situ</Emphasis> detection and identification of microorganisms at single-colony resolution by spectral imaging

The in situ detection and identification of environmental microorganisms is important for general microbial ecology research. In addition, the rapid detection of microbial contamination in food-processing plants is essential. The current paper proposes a method for the rapid detection and identification of microorganisms using spectral imaging. Photosynthetic and non-photosynthetic bacterial colonies with different absorption spectra in the near-infrared wavelength region were measured directly from a Petri dish. The bacterial colonies were distinguished and subsequently typed using multiple discriminant analysis. The accuracy of the detection and identification of colonies of various sizes was evaluated. The results showed that colonies with diameters of 100 and 300 μm could be detected and identified, respectively, with adequate accuracy. Moreover, using our novel application of this spectral-imaging technique, the bacterial detection and identification times were reduced by more than half and by a few weeks, respectively, compared with conventional methods.     

Fluorescence Assay Based on Aptamer-Quantum Dot Binding to <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Spores

A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.     

Steady State and Time-Resolved Fluorescence Studies of a Hemagglutinin from Moringa oleifera

The saccharide binding and conformational characterization of a hemagglutinin, a low molecular weight protein from the seeds of Moringa oleifera was studied using steady state and time resolved fluorescence. The lectin binds sugars LacNAc (K _a = 1380 M⁻¹) and fructose (K _a = 975 M⁻¹), as determined by the fluorescence spectroscopy. It has a single tryptophan per monomer which is exposed on the surface and is in a strong electropositive environment as revealed by quenching with iodide. Quenching of the fluorescence by acrylamide involved both static (K _s = 0.216 M⁻¹) and collisional (K _sv = 8.19 M⁻¹) components. The native protein showed two different lifetimes, τ ₁ (1.6 ns) and τ ₂ (4.36 ns) which decrease and get converted into a single one, (2.21 ns) after quenching with 0.15 M acrylamide. The bimolecular quenching constant, k _q was 7.55 × 10¹¹ M⁻¹ s⁻¹. ANS binding studies showed that the native protein has exposed hydrophobic patches which get further exposed at extreme acidic or alkaline pH. However, they get buried in the interior of the protein in presence of 1 M GdnHCl or urea.     

Flow cytometric discrimination of phytoplankton classes by fluorescence emission and excitation properties

The ataxonomic discrimination of phytoplankton species on the basis of fluorescence data obtained by multiwavelength excitation in combination with wavelength selective detection in flow cytometry is demonstrated. The discrimination is based on differences in pigment composition between the species, which are reflected in their spectral characteristics. Classification can be done both by making use of the absolute fluorescence intensities and with fluorescence parameter ratios. The latter approach has the advantage that size-related effects and instrument fluctuations are reduced to a large extent. Photoadaptation does influence the absolute as well as the ratioed parameters that are obtained but does not impede the classification into major ataxonomic groups.     

Fluorescence Characterization of the Hydrophobic Pocket of Cyclophilin B

Human cyclophilin B is a monomeric protein that contains two tryptophan residues, Trp104 and 128. Trp128-residue belongs to the binding site of cyclosporin A and is the homologous of Trp 121 in CyPA, while Trp104 residue belongs to the hydrophobic pocket. In the present work, we studied the dynamics of Trp residue(s) of cyclophilin B and of the CyPB_w128A mutant and of TNS-mutant complex. Our results showed that Trp-104 and TNS show restricted motions within their environments and that energy transfer between the two fluorophores is occurring.     

In VivoLactate Editing with Simultaneous Detection of Choline, Creatine, NAA, and Lipid Singlets at 1.5 T Using PRESS Excitation with Applications to the Study of Brain and Head and Neck Tumors

Two T2-independentJ-difference lactate editing schemes for the PRESS magnetic resonance spectroscopy localization sequence are introduced. The techniques, which allow for simultaneous acquisition of the lactate doublet (1.3 ppm) and edited singlets upfield of and including choline (3.2 ppm), exploit the dependence of the in-phase intensity of the methyl doublet upon the time interval separating two inversion (BASING) pulses applied to its coupling partner after initial excitation. Editing method 1, which allows for echo times TE =n/J(n= 1, 2, 3, …), alters the BASING carrier frequency for each of two cycles so that, for one cycle, the quartet is inverted, whereas, for the other cycle, the quartet is unaffected. Method 2, which also provides water suppression, allows for editing for TE > 1/Jby alternating, between cycles, the time interval separating the inversion pulses. Experimental results were obtained at 1.5 T using a Shinnar Le–Roux-designed maximum phase inversion pulse with a filter transition bandwidth of 55 Hz. Spectra were acquired from phantoms andin vivofrom the human brain and neck. In a neck muscle study, the lipid suppression factor, achieved partly through the use of a novel phase regularization algorithm, was measured to be over 10³. Spectra acquired from a primary brain and a metastatic neck tumor demonstrated the presence of lactate and choline signals consistent with abnormal spectral patterns. The advantages and limitations of the methods are analyzed theoretically and experimentally, and significance of the results is discussed.     

In VivoLongitudinally Detected ESR Measurements at Microwave Regions of 300, 700, and 900 MHz in Rats Treated with a Nitroxide Radical

A signal detector of longitudinally detected ESR (LODESR) is independent of the resonant frequency. We developed anin vivoLODESR spectrometer operating in the regions of 300, 700, and 900 MHz. Using this apparatus, we estimated signal intensities at different operating frequencies obtained from non- or high-dielectric loss phantoms that contained nitroxide radical solutions and from live rats that had received a nitroxide radical. Our result, higher signal intensities in the high-dielectric loss samples (such as physiological saline solution and animals) at a lower frequency, shows that the influence of a decrease in dielectric loss dominates over the signal reduction caused by smaller Zeeman splitting. We believe that this finding strongly supports anin vivoESR resonant frequency that tends to be low.