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Fluorescence Data (fluorescence + data)
Selected AbstractsClinical applications of laser scanning cytometryCYTOMETRY, Issue 3 2002Attila Tárnok Abstract This study reviews existing and potential clinical applications of laser scanning cytometry (LSC) and outlines possible future developments. LSC provides a technology for solid phase cytometry. Fluorochrome-labeled specimens are immobilized on microscopic slides that are placed on a conventional epifluorescence microscope and analyzed by one or two lasers. Data comparable to flow cytometry are generated. In addition, the position of each event is recorded, a feature that allows relocalization and visualization of each measured event. The major advantage of LSC compared with other cytometric methods is the combination of two features: (a) the minimal clinical sample volume needed and (b) the connection of fluorescence data and morphological information for the measured event. Since the introduction of LSC, numerous methods have been established for the analysis of cells, cellular compartments, and tissues. Although most cytometric methods use only two or three colors, the characterization of specimens with up to five fluorochromes is possible. Most clinical applications have been designed to determine ploidy and immunophenotype; other applications include analyses of tissue biopsies and sections, fluorescence in situ hybridization, and the combination of vital and nonvital information on a single-cell basis. With the currently available assays, LSC has proven its wide spectrum of clinical applicability in slide-based cytometry and can be introduced as a standard technology in multiple clinical settings. Cytometry (Clin. Cytometry) 50:133,143, 2002. © 2002 Wiley-Liss, Inc. [source] Fluorescent proteins for single-molecule fluorescence applicationsJOURNAL OF BIOPHOTONICS, Issue 1 2008Britta Seefeldt Abstract We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] The differentiation of biodegradable and non-biodegradable dissolved organic matter in wastewaters using fluorescence spectroscopyJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2002M Reynolds Abstract The chemical and biochemical oxygen demand values of a number of synthetic and wastewater samples were determined using fluorescence spectroscopy. Treated and untreated sewage samples were obtained from a local sewage treatment works while synthetic samples were analysed before, during, and after treatment via a rotating biodisc contactor. Fluorescence intensities were normalised using the water Raman signal as an internal standard and corrections applied to take into account the attenuation effects caused by the sample matrix. The fluorescence emission spectra (,exc,=,280,nm) of synthetic and sewage samples were very similar in that two main fluorescence bands centred around 350,nm and 440,nm were observed in all samples. Normalised fluorescence data, centred at 350,nm, correlate well with corresponding BOD, COD and TOC values (R2 values ranging between 0.93 and 0.98). Using BOD, COD and TOC data the fluorescence at 350,nm and 440,nm can be apportioned to biodegradable and non-biodegradable dissolved organic matter respectively. The findings of this research show that fluorescence data can be used to quantify oxygen demand values (chemical and biochemical) and total organic carbon values. Furthermore, the fluorescence spectral response can be apportioned to biodegradable (BOD) and non-biodegradable (COD,,,BOD) dissolved organic matter. The potential of using fluorescence spectroscopy as a possible tool for real-time monitoring of sewage wastes is discussed. © 2002 Society of Chemical Industry [source] A fully robust PARAFAC method for analyzing fluorescence dataJOURNAL OF CHEMOMETRICS, Issue 3 2009Sanne Engelen Abstract Parallel factor analysis (PARAFAC) is a widespread method for modeling fluorescence data by means of an alternating least squares procedure. Consequently, the PARAFAC estimates are highly influenced by outlying excitation,emission landscapes (EEM) and element-wise outliers, like for example Raman and Rayleigh scatter. Recently, a robust PARAFAC method that circumvents the harmful effects of outlying samples has been developed. For removing the scatter effects on the final PARAFAC model, different techniques exist. Newly, an automated scatter identification tool has been constructed. However, there still exists no robust method for handling fluorescence data encountering both outlying EEM landscapes and scatter. In this paper, we present an iterative algorithm where the robust PARAFAC method and the scatter identification tool are alternately performed. A fully automated robust PARAFAC method is obtained in that way. The method is assessed by means of simulations and a laboratory-made data set. Copyright © 2009 John Wiley & Sons, Ltd. [source] Handling of Rayleigh and Raman scatter for PARAFAC modeling of fluorescence data using interpolationJOURNAL OF CHEMOMETRICS, Issue 3-4 2006Morteza Bahram Abstract Fluorescence excitation-emission matrix (EEM) measurements are useful in fields such as food science, analytical chemistry, biochemistry and environmental science. EEMs contain information which can be modeled using the parallel factor analysis (PARAFAC) model but the data analysis is often complicated due to both Rayleigh and Raman scattering. There are several established ways to deal with scattering effects. However, all of these methods have associated problems. This paper develops a new method for handling scattering using interpolation in the areas affected by first- and second-order Rayleigh and Raman scatter in such a way that the interfering signal is, at best, removed. The suggested method is fast and requires no additional input other than specifying the scattering region. The results of the proposed method were compared with those obtained from common alternative approaches used for preprocessing fluorescence data before analysis with PARAFAC and were shown to be equally good for various types of EEM data. The main advantage of the interpolation method is in its lack of additional metaparameters, its algorithmic speed and subsequent speed-up of PARAFAC modeling. It also allows for using EEM data in software not able to handle missing data. Copyright © 2007 John Wiley & Sons, Ltd. [source] Practical aspects of PARAFAC modeling of fluorescence excitation-emission dataJOURNAL OF CHEMOMETRICS, Issue 4 2003C. M. Andersen Abstract This paper presents a dedicated investigation and practical description of how to apply PARAFAC modeling to complicated fluorescence excitation,emission measurements. The steps involved in finding the optimal PARAFAC model are described in detail based on the characteristics of fluorescence data. These steps include choosing the right number of components, handling problems with missing values and scatter, detecting variables influenced by noise and identifying outliers. Various validation methods are applied in order to ensure that the optimal model has been found and several common data-specific problems and their solutions are explained. Finally, interpretations of the specific models are given. The paper can be used as a tutorial for investigating fluorescence landscapes with multi-way analysis. Copyright © 2003 John Wiley & Sons, Ltd. [source] Anthrax vaccine powder formulations for nasal mucosal deliveryJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2006Ge Jiang Abstract Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6,8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7,8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:80,96, 2006 [source] Photophysics in Motionally constrained Bioenvironment: Interactions of Norharmane with Bovine Serum Albumin,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2005Arabinda Mallick ABSTRACT Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (,G) for the probe-protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water-dioxane mixture with varying composition. [source] Spectroscopic Properties of Various Quinolone Antibiotics in Aqueous,organic Solvent Mixtures¶PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2004Hyoung-Ryun Park The spectroscopic properties of enoxacin (ENO), oxolinic acid (OXO) and nalidixic acid (NAL) were studied in various H2O-CH3OH and H2O-CH3CN mixed solvents because these solvents were thought to behave as a biological mimetic system. ENO has piperazinyl group, but OXO and NAL do not have this substituent. The fluorescence emission spectra of ENO were very sensitive to the composition of the solvents. In the Lippert-Mataga analysis of the steady-state fluorescence data, clear reverse solvatochromism was exhibited for ENO in both mixed solvents. This observation can be explained using the excited state twisted intramolecular charge transfer (TICT) from the nitrogen of the piperazinyl group to the keto oxygen. Theoretical calculations further support this observation. The nonradiative and radiative rate constants of these molecules were analyzed as a function of dipolarity,polarizability (,*) and hydrogen bond donor acidity (,) of the mixed solvents. These results for ENO were consistent with the suggested mechanism of the TICT very well. The influence of bulk dielectric effect was more significant relative to the specific hydrogen bonding interactions. The emission spectra of OXO and NAL do not exhibit any characteristic responses to the properties of the solvent. [source] Substrate interactions of the electroneutral Na+ -coupled inorganic phosphate cotransporter (NaPi-IIc)THE JOURNAL OF PHYSIOLOGY, Issue 17 2009Chiara Ghezzi The SLC34 solute carrier family comprises the electrogenic NaPi-IIa/b and the electroneutral NaPi-IIc, which display Na+ : Pi cotransport stoichiometries of 3 : 1 and 2 : 1, respectively. We previously proposed that NaPi-IIc lacks one of the three Na+ interaction sites hypothesised for the electrogenic isoforms, but, unlike NaPi-IIa/b, its substrate binding order is undetermined. By expressing NaPi-IIc in Xenopus oocytes, isotope influx and efflux assays gave results consistent with Na+ being the first and last substrate to bind. To further investigate substrate interactions, we applied a fluorometry-based technique that uses site-specific labelling with a fluorophore to characterize substrate-induced conformational changes. A novel Cys was introduced in the third extracellular loop of NaPi-IIc that could be labelled with a reporter fluorophore (MTS-TAMRA). Although labelling resulted in suppression of cotransport as previously reported for the electrogenic isoforms, changes in fluorescence were induced by changes in extracellular Na+ concentration in the absence of Pi and by changes in extracellular Pi concentration in presence of Na+. These data, combined with 32P uptake data, also support a binding scheme in which Na+ is the first substrate to interact. Moreover, the apparent Pi affinity from fluorometry agreed with that from 32P uptake, confirming the applicability of the fluorometric technique for kinetic studies of electroneutral carriers. Analysis of the fluorescence data showed that like the electrogenic NaPi-IIb, 2 Na+ ions interact cooperatively with NaPi-IIc before Pi binding, which implies that only one of these is translocated. This result provides compelling evidence that SLC34 proteins share common motifs for substrate interaction and that cotransport and substrate binding stoichiometries are not necessarily equivalent. [source] Controlled enzymatic removal of damaging casein layers on medieval wall paintingsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2002Sascha Beutel Abstract A new, gentle enzymatic method was developed for a controlled removal of casein layers from medieval wall paintings. These casein layers were applied over the last 60 years on wall paintings in order to decrease substantial damage due to a peeling off of the frescoes from the roughcast surface due to environmental effects. However, due to the aging of the casein layers (at 40,50 years), a more drastic peeling occurred and the danger of total destruction of the wall paintings is severe. Thus, screening was performed to find the most suitable enzyme for casein digestion. Alcalase 2.5 DX L was the most appropriate enzyme for an effective proteolysis reaction. The enzyme was immobilized on functionalized cellulose membrane. A membrane pad system with immobilized enzymes was developed which could be pressed on the casein layers on the wall painting. A controlled removal of the casein layers by proteolytic digestion was observed and it was possible to continuously wash off the hydrolyzed casein fragments from the wall painting surface by an aqueous carbonate buffer flowing through the membrane pad. The removal and the digestion was monitored by reverse HPLC. Additionally, an on-line monitoring system was set up in order to continuously follow the casein layer removal and the digestion procedure directly on the wall painting. This technique is based on noninvasive 2D-fluorescence monitoring. Optical fiber systems were used to continuously monitor the fluorescence intensity of casein-bound tryptophan. The off-line data were verified with the on-line 2D-fluorescence data. Based on the scientific result an appropriate technique for the controlled enzymatic removal of damaging casein layers on the surface of medieval wall paintings using immobilized enzyme is now available. It is now applied to remove such casein layers from medieval wall paintings in the Allerheiligen-Kapelle Cloister, Wienhausen, Germany, and the St. Alexander Kirche, Wildeshausen, Germany. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 13,21, 2002. [source] Bispectral fluorescence imaging of aggressive basal cell carcinoma combined with histopathological mapping: a preliminary study indicating a possible adjunct to Mohs micrographic surgeryBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2006B. Stenquist Summary Background, Fluorescence imaging is an attractive diagnostic technique for skin tumour demarcation with potential to move to clinical use. Bispectral fluorescence imaging combines skin autofluorescence with ,-aminolaevulinic acid-induced fluorescence. To evaluate the technique, fluorescence data must be compared with the histopathological extent of the tumour, which is the purpose of the current study. Objectives, To investigate the agreement between bispectral fluorescence images and the histopathological tumour boundary of ill-defined basal cell carcinomas (BCCs). After fluorescence imaging the tumours were removed using Mohs micrographic surgery (MMS) to obtain histopathological maps of the tumour boundaries. Methods, Twelve patients with aggressive BCC of mean diameter 16 mm (range 5,32) in the face were included in the study. The patients were subjected to bispectral fluorescence imaging within the 2 months prior to MMS. The fluorescence images and histopathological maps were aligned using image warping. Results, Five patients (42%) showed good agreement with the histopathological mapping and the remaining seven patients (58%) showed partial agreement. Bispectral investigation combining autofluorescence with protoporphyrin IX (PpIX) fluorescence generally yielded better agreement with the histopathological boundaries of the tumours compared with using only the PpIX fluorescence. Conclusions, In this preliminary study the fluorescence has been compared with the histopathological tumour boundaries. The result implies that the technique can be applied as a useful tool for indicating tumour boundary of aggressive BCCs. Further refinement is needed to be able to indicate the exact tumour border. [source] Supramolecular Assembly of 2,7-Dimethyldiazapyrenium and Cucurbit[8]uril: A New Fluorescent Host for Detection of Catechol and DopamineCHEMISTRY - A EUROPEAN JOURNAL, Issue 23 2005Vladimir Sindelar Dr. Abstract The formation of a highly stable inclusion complex between 2,7-dimethyldiazapyrenium (Me2DAP2+) and the cucurbit[8]uril host (CB8) was demonstrated by X-ray crystallography; MALDI-TOF mass spectrometry; and 1H NMR, electronic absorption, and emission spectroscopy. The equilibrium association constant was determined to be 8.9(±0.2)×105 L,mol,1 from UV-visible data and 8.4(±1.5)×105 L,mol,1 from fluorescence data. The Me2DAP2+,CB8 inclusion complex acted as a host to bind compounds containing aromatic ,-donor moieties (D), such as catechol and dopamine. This point was demonstrated by 1H NMR spectroscopy, and electrochemical and emission measurements. Fluorescence detection of the Me2DAP2+,D,CB8 ternary complexes was evident in aqueous solution and on the surface of silica particles, to which fluorescent diazapyrenium units had been covalently immobilized. [source] Eosinophilia in nasal polyposis: its objective quantification and clinical relevanceCLINICAL & EXPERIMENTAL ALLERGY, Issue 1 2004A. O. H. Gerstner Summary Background Eosinophilia within nasal polyps is often taken as a criterion for adjuvant medical treatment postoperatively such as topical steroids. Objective This study was performed in order to validate a new technique for objective quantification of eosinophilia by using laser scanning cytometry (LSC), to compare these results with manual scoring and routine histopathology, and to correlate them with the history of allergy or recurrence. Methods LSC was used for semi-automated analysis of single-cell preparations from representative ethmoidal polyps obtained during routine paranasal sinus surgery (n=41). This microscope-based instrument scans the cells after immobilization of cells on a glass slide and after triple staining of cytokeratin, eosinophilic granula, and DNA. The location of each cell is stored with the fluorescence data. Therefore, the morphology of every cell can be documented by re-staining with haemotoxylin and eosin and re-localization on the slide. Subsequently, slides were subjected to manual scoring. The remaining polyps were analysed by routine histopathology. Results Data from LSC and manual scoring showed good correlation (r=0.81, P<0.001), whereas there were discrepancies with histopathology. Eosinophilia scored by LSC and histopathology was neither correlated with the history of allergy nor with recurrence as determined by Fisher's exact test independent of the definition of eosinophilia (2%, 3%, or 5% of all cells). Conclusion Scoring eosinophilia by LSC in comparison with histopathology does not contribute to a more reliable basis for adjuvant medical therapy in nasal polyposis. Instead, functional parameters (cytokine production, apoptosis) may serve better. [source] | ||||||||||||||||