The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions.

PubMed

Žoldák, Gabriel; Jancura, Daniel; Sedlák, Erik

2017-06-01

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady-state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high-throughput drug screening campaigns. © 2017 The Protein Society.

Intensity and pressure dependence of resonance fluorescence of OH induced by a tunable UV laser

NASA Technical Reports Server (NTRS)

Killinger, D. K.; Wang, C. C.; Hanabusa, M.

1976-01-01

The intensity and pressure dependence of the fluorescence spectrum of OH in the presence of N2 and H2O molecules was studied. Saturation of the absorption transition was observed at low pressures, and the corresponding fluorescence signal was found to vary as the square root of the exciting intensity. This observed dependence agreed with the predicted dependence which took into account the presence of laser modes in the spectrum of the exciting radiation. With full laser power incident, a saturation parameter as high as 3 x 10 to the 5th was observed. The fluorescence spectrum was found to peak at 3145 and at 3090 A, with the relative peak intensities dependent upon gas pressures and upon the particular rotational electronic transition used for excitation. It is concluded that vibrational relaxation of the electronically excited OH due to water vapor in the system plays a dominant role in determining the observed fluorescence spectrum.

A molecular-sized optical logic circuit for digital modulation of a fluorescence signal

NASA Astrophysics Data System (ADS)

Nishimura, Takahiro; Tsuchida, Karin; Ogura, Yusuke; Tanida, Jun

2018-03-01

Fluorescence measurement allows simultaneous detection of multiple molecular species by using spectrally distinct fluorescence probes. However, due to the broad spectra of fluorescence emission, the multiplicity of fluorescence measurement is generally limited. To overcome this limitation, we propose a method to digitally modulate fluorescence output signals with a molecular-sized optical logic circuit by using optical control of fluorescence resonance energy transfer (FRET). The circuit receives a set of optical inputs represented with different light wavelengths, and then it switches high and low fluorescence intensity from a reporting molecule according to the result of the logic operation. By using combinational optical inputs in readout of fluorescence signals, the number of biomolecular species that can be identified is increased. To implement the FRET-based circuits, we designed two types of basic elements, YES and NOT switches. An YES switch produces a high-level output intensity when receiving a designated light wavelength input and a low-level intensity without the light irradiation. A NOT switch operates inversely to the YES switch. In experiments, we investigated the operation of the YES and NOT switches that receive a 532-nm light input and modulate the fluorescence intensity of Alexa Fluor 488. The experimental result demonstrates that the switches can modulate fluorescence signals according to the optical input.

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase.

PubMed

Zhang, Chong; Liu, Min-Sheng; Xing, Xin-Hui

2009-09-01

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60 degrees C in either single or fusion form can be completely recovered by treatment at 0 degrees C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (Rgfp/Axyl) at 15 degrees C and 37 degrees C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the Rgfp/Axyl ratio at 15 degrees C is ten times of that at 37 degrees C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.

Velocity landscape correlation resolves multiple flowing protein populations from fluorescence image time series.

PubMed

Pandžić, Elvis; Abu-Arish, Asmahan; Whan, Renee M; Hanrahan, John W; Wiseman, Paul W

2018-02-16

Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Fluorescent biosensor for the detection of hyaluronidase: intensity-based ratiometric sensing and fluorescence lifetime-based sensing using a long lifetime azadioxatriangulenium (ADOTA) fluorophore.

PubMed

Chib, Rahul; Mummert, Mark; Bora, Ilkay; Laursen, Bo W; Shah, Sunil; Pendry, Robert; Gryczynski, Ignacy; Borejdo, Julian; Gryczynski, Zygmunt; Fudala, Rafal

2016-05-01

In this report, we have designed a rapid and sensitive, intensity-based ratiometric sensing as well as lifetime-based sensing probe for the detection of hyaluronidase activity. Hyaluronidase expression is known to be upregulated in various pathological conditions. We have developed a fluorescent probe by heavy labeling of hyaluronic acid with a new orange/red-emitting organic azadioxatriangulenium (ADOTA) fluorophore, which exhibits a long fluorescence lifetime (∼20 ns). The ADOTA fluorophore in water has a peak fluorescence lifetime of ∼20 ns and emission spectra centered at 560 nm. The heavily ADOTA-labeled hyaluronic acid (HA-ADOTA) shows a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal, and a shorter fluorescence lifetime (∼4 ns) due to efficient self-quenching and formation of aggregates. In the presence of hyaluronidase, the brightness and fluorescence lifetime of the sample increase with a blue shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA-ADOTA probe at 560 and 605 nm can be used as the sensing method for the detection of hyaluronidase. The cleavage of the hyaluronic acid macromolecule reduces the energy migration between ADOTA molecules, as well as the degree of self-quenching and aggregation. This probe can be efficiently used for both intensity-based ratiometric sensing as well as fluorescence lifetime-based sensing of hyaluronidase. The proposed method makes it a rapid and sensitive assay, useful for analyzing levels of hyaluronidase in relevant clinical samples like urine or plasma. Graphical Abstract Scheme showing cleavage of HA-ADOTA probe by hyaluronidase and the change in the emission spectrum of HA-ADOTA probe before and after cleavage by hyaluronidase.

Sub-annual paleoenvironmental information evaluated from intensity variations of fluorescent annual layers in a stalagmite from Ryuo-do Cave, Nagasaki Prefecture, western Japan

NASA Astrophysics Data System (ADS)

Sasaki, Hana; Onishi, Yuri; Ishihara, Yoshiro; Yoshimura, Kazuhisa

2017-04-01

vary stratigraphically, and multiple types of fluorescence intensity pattern are observed in the stalagmite. When the co-precipitation of FA is governed by the hiatus model, it is suggested that a gradual increase in the annual layers will result from a large accumulation of calcite after the annual peak in the FA concentration, whereas there will be a gradual decrease if the main growth occurs before the annual peak in FA concentration. However, in the case of the PC model, a gradually increasing type of pattern is formed if the main growth occurs before the annual peak in FA concentration, and a gradually decreasing type is formed if the main growth occurs afterwards. If the annual peak of FA concentration occurs several months after high summer, it is suggested that intervals showing a gradually increasing type were formed in winter, and intervals showing a gradually decreasing type were formed in the early summer, in the case of the hiatus model. In the case of PC model, the seasons are reversed. In the climatic environment around the Ryuo-do Cave, the growth rates of stalagmites are affected by cave air circulation in winter and by rainfall (rainy season) in early summer.

A Semi-Empirical Formula of the Dependence of the Fluorescence Intensity of Naphthalene on Temperature and the Oxygen Concentration

NASA Astrophysics Data System (ADS)

An, B.; Wang, Z.-G.; Yang, L.-C.; Li, X.-P.

2017-09-01

Two-ring aromatics, such as naphthalene, are important fluorescent components of kerosene in the planar laser-induced fluorescent (PLIF) technique. Quantifying measurements of kerosene vapor concentrations by PLIF require a prior knowledge of the fluorescence intensity of naphthalene over a wide temperature and oxygen concentration range. To promote the application of PLIF, a semi-empirical formula based on the collision theory and experimental data at the laser wavelength of 266 nm and a pressure of 0.1 MPa is established to predict the fluorescence intensity of naphthalene at different temperatures and oxygen concentrations. This formula takes vibrational states, temperature, and oxygen quenching into account. Verified by published experimental data, the formula can predict the fluorescence intensity of naphthalene with an error less than 9%.

Enhancing fluorescence intensity of Ellagic acid in Borax-HCl-CTAB micelles

NASA Astrophysics Data System (ADS)

Wang, Feng; Huang, Wei; Zhang, Shuai; Liu, Guokui; Li, Kexiang; Tang, Bo

2011-03-01

Ellagic acid (C 14H 6O 8), a naturally occurring phytochemical, found mainly in berries and some nuts, has anticarcinogenic and antioxidant properties. It is found that fluorescence of Ellagic acid (EA) is greatly enhanced by micelle of cetyltrimethylammonium bromide (CTAB) surfactant. Based on this effect, a sensitive proposed fluorimetric method was applied for the determination of Ellagic acid in aqueous solution. In the Borax-HCl buffer, the fluorescence intensity of Ellagic acid in the presence of CTAB is proportional to the concentration of Ellagic acid in range from 8.0 × 10 -10 to 4.0 × 10 -5 mol L -1; and the detection limits are 3.2 × 10 -10 mol L -1 and 5.9 × 10 -10 mol L -1 excited at 266 nm and 388 nm, respectively. The actual samples of pomegranate rinds are simply manipulated and satisfactorily determined. The interaction mechanism studies argue that the negative EA-Borax complex is formed and solubilized in the cationic surfactant CTAB micelle in this system. The fluorescence intensity of EA enhances because the CTAB micelle provides a hydrophobic microenvironment for EA-Borax complex, which can prevent collision with water molecules and decrease the energy loss of EA-Borax complex.

Label-Free Fluorescent DNA Dendrimers for microRNA Detection Based On Nonlinear Hybridization Chain Reaction-Mediated Multiple G-Quadruplex with Low Background Signal.

PubMed

Xue, Qingwang; Liu, Chunxue; Li, Xia; Dai, Li; Wang, Huaisheng

2018-04-18

Various fluorescent sensing systems for miRNA detection have been developed, but they mostly contain enzymatic amplification reactions and label procedures. The strict reaction conditions of tool enzymes and the high cost of labeling limit their potential applications, especially in complex biological matrices. Here, we have addressed the difficult problems and report a strategy for label-free fluorescent DNA dendrimers based on enzyme-free nonlinear hybridization chain reaction (HCR)-mediated multiple G-quadruplex for simple, sensitive, and selective detection of miRNAs with low-background signal. In the strategy, a split G-quadruplex (3:1) sequence is ingeniously designed at both ends of two double-stranded DNAs, which is exploited as building blocks for nonlinear HCR assembly, thereby acquiring a low background signal. A hairpin switch probe (HSP) was employed as recognition and transduction element. Upon sensing the target miRNA, the nonlinear HCR assembly of two blocks (blocks-A and blocks-B) was initiated with the help of two single-stranded DNA assistants, resulting in chain-branching growth of DNA dendrimers with multiple G-quadruplex incorporation. With the zinc(II)-protoporphyrin IX (ZnPPIX) selectively intercalated into the multiple G-quadruplexes, fluorescent DNA dendrimers were obtained, leading to an exponential fluorescence intensity increase. Benefiting from excellent performances of nonlinear HCR and low background signal, this strategy possesses the characteristics of a simplified reaction operation process, as well as high sensitivity. Moreover, the proposed fluorescent sensing strategy also shows preferable selectivity, and can be implemented without modified DNA blocks. Importantly, the strategy has also been tested for miRNA quantification with high confidence in breast cancer cells. Thus, this proposed strategy for label-free fluorescent DNA dendrimers based on a nonlinear HCR-mediated multiple G-quadruplex will be turned into an alternative

Fluorescence multispectral imaging-based diagnostic system for atherosclerosis.

PubMed

Ho, Cassandra Su Lyn; Horiuchi, Toshikatsu; Taniguchi, Hiroaki; Umetsu, Araya; Hagisawa, Kohsuke; Iwaya, Keiichi; Nakai, Kanji; Azmi, Amalina; Zulaziz, Natasha; Azhim, Azran; Shinomiya, Nariyoshi; Morimoto, Yuji

2016-08-20

Composition of atherosclerotic arterial walls is rich in lipids such as cholesterol, unlike normal arterial walls. In this study, we aimed to utilize this difference to diagnose atherosclerosis via multispectral fluorescence imaging, which allows for identification of fluorescence originating from the substance in the arterial wall. The inner surface of extracted arteries (rabbit abdominal aorta, human coronary artery) was illuminated by 405 nm excitation light and multispectral fluorescence images were obtained. Pathological examination of human coronary artery samples were carried out and thickness of arteries were calculated by measuring combined media and intima thickness. The fluorescence spectra in atherosclerotic sites were different from those in normal sites. Multiple regions of interest (ROI) were selected within each sample and a ratio between two fluorescence intensity differences (where each intensity difference is calculated between an identifier wavelength and a base wavelength) from each ROI was determined, allowing for discrimination of atherosclerotic sites. Fluorescence intensity and thickness of artery were found to be significantly correlated. These results indicate that multispectral fluorescence imaging provides qualitative and quantitative evaluations of atherosclerosis and is therefore a viable method of diagnosing the disease.

Amphiphilic inclusion spaces for various guests and regulation of fluorescence intensity of 1,8-bis(4-aminophenyl)anthracene crystals.

PubMed

Sugino, Misa; Hatanaka, Keisuke; Araki, Yusuke; Hisaki, Ichiro; Miyata, Mikiji; Tohnai, Norimitsu

2014-03-10

A host framework for inclusion of various guest molecules was investigated by preparation of inclusion crystals of 1,8-bis(4-aminophenyl)anthracene (1,8-BAPA) with organic solvents. X-ray crystallographic analysis revealed construction of the same inclusion space incorporating 1,8-BAPA and eight guest molecules including both non-polar (benzene) and polar guests (N,N-dimethylformamide, DMF). Fluorescence efficiencies varied depending on guest molecule polarity; DMF inclusion crystals exhibited the highest fluorescence intensity (ΦF=0.40), four times as high as that of a benzene inclusion crystal (ΦF=0.10). According to systematic investigations of inclusion phenomena, strong host–guest interactions and filling of the inclusion space led to a high fluorescence intensity. Temperature-dependent fluorescence spectral measurements revealed these factors effectively immobilised the host framework. Although hydrogen bonding commonly decreases fluorescence intensity, the present study demonstrated that such strong interactions provide excellent conditions for fluorescence enhancement. Thus, this remarkable behaviour has potential application toward sensing of highly polar molecules, such as biogenic compounds.

High intensity portable fluorescent light

NASA Technical Reports Server (NTRS)

Kendall, F. B.

1972-01-01

Eight high intensity portable fluorescent lights were produced. Three prototype lights were also produced, two of which were subsequently updated to the physical and operational configuration of the qualification and flight units. Positioning of lamp apertures and reflectors in these lights is such that the light is concentrated and intensified in a specific pattern rather than widely diffused. Indium amalgam control of mercury vapor pressure in the lamp gives high output at lamp ambient temperatures up to 105 C. A small amount of amalgam applied to each electrode stem helps to obtain fast warm-up. Shrinking a Teflon sleeve on the tube and potting metal caps on each end of the lamp minimizes dispersion of mercury vapor and glass particles in the event of accidental lamp breakage. Operation at 20 kHz allows the lamps to consume more power than at low frequency, thus increasing their light output and raising their efficiency. When used to expose color photographic film, light from the lamps produces results approximately equal to sunlight.

Fluorescence Intensity- and Lifetime-Based Glucose Sensing Using Glucose/Galactose-Binding Protein

PubMed Central

Pickup, John C.; Khan, Faaizah; Zhi, Zheng-Liang; Coulter, Jonathan; Birch, David J. S.

2013-01-01

We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested.Fluorescence technique is one of the major solutions for achieving the continuous and noninvasive glucose sensor for diabetes. In this article, a highly sensitive nanostructured sensor is developed to detect extremely small amounts of aqueous glucose by applying fluorescence energy transfer (FRET). A one-pot method is applied to produce the dextran-fluorescein isothiocyanate (FITC)-conjugating mesoporous silica nanoparticles (MSNs), which afterward interact with the tetramethylrhodamine isothiocyanate (TRITC)-labeled concanavalin A (Con A) to form the FRET nanoparticles (FITC-dextran-Con A-TRITC@MSNs). The nanostructured glucose sensor is then formed via the self-assembly of the FRET nanoparticles on a transparent, flexible, and biocompatible substrate, e.g., poly(dimethylsiloxane). Our results indicate the diameter of the MSNs is 60 ± 5 nm. The difference in the images before and after adding 20 μl of glucose (0.10 mmol/liter) on the FRET sensor can be detected in less than 2 min by the laser confocal laser scanning microscope. The correlation between the ratio of fluorescence intensity, I(donor)/I(acceptor), of the FRET sensor and the concentration of aqueous glucose in the range of 0.04–4 mmol/liter has been investigated; a linear relationship is found. Furthermore, the durability of the nanostructured FRET sensor is evaluated for 5 days. In addition, the recorded images can be converted to digital images by

Nanostructured fluorescent particles for glucose sensing

NASA Astrophysics Data System (ADS)

Grant, Patrick S.; Fang, Ming; Lvov, Yuri; McShane, Michael J.

2002-05-01

Self-assembled thin films containing embedded enzymes and fluorescent indicators are being developed for use as highly specific glucose biosensors. The sensors are fabricated using electrostatic Layer-by-Layer (LBL) adsorption to create oxygen-sensitive (Ruthenium-based) layers, the fluorescent intensity of which responds to changes in local oxygen levels. Oxygen is consumed locally by the reaction between glucose oxidase (GOx) molecules and glucose. Latex particles serve as the templates for our sensors and fabrication is carried out through the alternate adsorption of multiple levels of {GOx/polycation} and {Ruthenium-polycation/polyanion} bilayers. Additional fluorescence layers as well as fluorescent latex are being considered as internal intensity references to allow ratiometric monitoring. Films adsorbed to the nanoparticle templates are being studied to understand the fundamental chemical and optical properties, including enzymatic activity, spectral shape and emission intensity. Enzymatic activity is retained and stability is improved after adsorption, and increased surface area afforded by the particles allows use of increased numbers of molecules. Fluorescence is also maintained, though blue shifts are observed in emission spectra, and indicator activity remains. In vitro characterization studies demonstrate the feasibility of the particles as glucose biosensors, and future work will aim to optimize the response for neural monitoring.

[Retrospective, descriptive, observational study of treatment of multiple actinic keratoses with topical methyl aminolevulinate and red light: results in clinical practice and correlation with fluorescence imaging].

PubMed

Fernández-Guarino, M; Harto, A; Sánchez-Ronco, M; Pérez-García, B; Marquet, A; Jaén, P

2008-12-01

Actinic keratosis (AK) is one of the most common skin diseases seen in clinical practice. In the last 5 years, several studies assessing the efficacy of photodynamic therapy in the treatment of multiple AKs have been published. We aimed to assess the clinical outcomes of photodynamic therapy in patients with multiple AKs and the correlation of those outcomes with fluorescence imaging. In this retrospective, descriptive, observational study of 57 patients treated in our hospital with photodynamic therapy for multiple AKs, we recorded age, sex, and lesion site (face, scalp, and dorsum of the hands). All patients were treated in the same way: methyl aminolevulinic acid (Metvix) was applied for 3 hours and the skin then irradiated with red light at 630 nm, 37 J/cm(2), for 7.5 minutes (Aktilite). The response, remission duration, tolerance, number of sessions, and fluorescence images were recorded by site. The chi(2) test was used to assess between-site differences and the correlation between fluorescence imaging and clinical response. The greatest improvements were obtained for facial lesions; these required fewer sessions and remission lasted longer than lesions at other sites. The treatment was best tolerated on the dorsum of the hands. The fluorescence area and the reduction in intensity on applying treatment were found to be strongly and significantly correlated with the extent of clinical response. Overall, the outcomes of treatment of multiple AKs with photodynamic therapy are better for the face than for the scalp and dorsum of the hands. Fluorescence imaging may be an effective tool for predicting response to treatment.

Chair-side detection of Prevotella Intermedia in mature dental plaque by its fluorescence.

PubMed

Nomura, Yoshiaki; Takeuchi, Hiroaki; Okamoto, Masaaki; Sogabe, Kaoru; Okada, Ayako; Hanada, Nobuhiro

2017-06-01

Prevotella intermedia/nigrescens is one of the well-known pathogens causing periodontal diseases, and the red florescence excited by the visible blue light caused by the protoporphyrin IX in the bacterial cells could be useful for the chair-side detection. The aim of this study was to evaluated levels of periodontal pathogen, especially P. intermedia in clinical samples of red fluorescent dental plaque. Thirty two supra gingival plaque samples from six individuals were measured its fluorescence at 640nm wavelength excited by 409nm. Periodontopathic bacteria were counted by the Invader PLUS PCR assay. Co-relations the fluorescence intensity and bacterial counts were analyzed by Person's correlation coefficient and simple and multiple regression analysis. Positive and negative predictive values of the fluorescence intensities for with or without P. intermedia in supragingival plaque was calculated. When relative fluorescence unit (RFU) were logarithmic transformed, statistically significant linear relations between RFU and bacterial counts were obtained for P. intermedia, Porphyromonas gingivalis and Tannerella forsythia. By the multiple regression analysis, only P. intermedia had statistically significant co-relation with fluorescence intensities. All of the fluorescent dental plaque contained P. intermedia m. In contrast, 28% of non-fluorescent plaques contained P. intermedia. To check the fluorescence dental plaque in the oral cavity could be the simple chair-side screening of the mature dental plaque before examining the periodontal pathogens especially P. intermedia by the PCR method. Copyright © 2017 Elsevier B.V. All rights reserved.

Photoactivatable fluorescent probes reveal heterogeneous nanoparticle permeation through biological gels at multiple scales

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schuster, Benjamin S.; Allan, Daniel B.; Kays, Joshua C.

Diffusion through biological gels is crucial for effective drug delivery using nanoparticles. Here, we demonstrate a new method to measure diffusivity over a large range of length scales – from tens of nanometers to tens of micrometers – using photoactivatable fluorescent nanoparticle probes. We have applied this method to investigate the length-scale dependent mobility of nanoparticles in fibrin gels and in sputum from patients with cystic fibrosis (CF). Nanoparticles composed of poly(lactic-co-glycolic acid), with polyethylene glycol coatings to resist bioadhesion, were internally labeled with caged rhodamine to make the particles photoactivatable. We activated particles within a region of sample usingmore » brief, targeted exposure to UV light, uncaging the rhodamine and causing the particles in that region to become fluorescent. We imaged the subsequent spatiotemporal evolution in fluorescence intensity and observed the collective particle diffusion over tens of minutes and tens of micrometers. We also performed complementary multiple particle tracking experiments on the same particles, extending significantly the range over which particle motion and its heterogeneity can be observed. In fibrin gels, both methods showed an immobile fraction of particles and a mobile fraction that diffused over all measured length scales. In the CF sputum, particle diffusion was spatially heterogeneous and locally anisotropic but nevertheless typically led to unbounded transport extending tens of micrometers within tens of minutes. Lastly, these findings provide insight into the mesoscale architecture of these gels and its role in setting their permeability on physiologically relevant length scales, pointing toward strategies for improving nanoparticle drug delivery.« less

Photoactivatable fluorescent probes reveal heterogeneous nanoparticle permeation through biological gels at multiple scales

DOE PAGES

Schuster, Benjamin S.; Allan, Daniel B.; Kays, Joshua C.; ...

2017-05-31

Diffusion through biological gels is crucial for effective drug delivery using nanoparticles. Here, we demonstrate a new method to measure diffusivity over a large range of length scales – from tens of nanometers to tens of micrometers – using photoactivatable fluorescent nanoparticle probes. We have applied this method to investigate the length-scale dependent mobility of nanoparticles in fibrin gels and in sputum from patients with cystic fibrosis (CF). Nanoparticles composed of poly(lactic-co-glycolic acid), with polyethylene glycol coatings to resist bioadhesion, were internally labeled with caged rhodamine to make the particles photoactivatable. We activated particles within a region of sample usingmore » brief, targeted exposure to UV light, uncaging the rhodamine and causing the particles in that region to become fluorescent. We imaged the subsequent spatiotemporal evolution in fluorescence intensity and observed the collective particle diffusion over tens of minutes and tens of micrometers. We also performed complementary multiple particle tracking experiments on the same particles, extending significantly the range over which particle motion and its heterogeneity can be observed. In fibrin gels, both methods showed an immobile fraction of particles and a mobile fraction that diffused over all measured length scales. In the CF sputum, particle diffusion was spatially heterogeneous and locally anisotropic but nevertheless typically led to unbounded transport extending tens of micrometers within tens of minutes. Lastly, these findings provide insight into the mesoscale architecture of these gels and its role in setting their permeability on physiologically relevant length scales, pointing toward strategies for improving nanoparticle drug delivery.« less

Fe II fluorescence and anomalous C IV doublet intensities in symbiotic novae

NASA Technical Reports Server (NTRS)

Michalitsianos, A. G.; Kafatos, M.; Meier, S. R.

1992-01-01

The variation of absolute intensities of Bowen-excited Fe II emission in the symbiotic stars RR Tel, RX Pup, and AG Peg is examined. The C IV doublet intensity ratios in RR Tel were not anomalous between 1979 and 1989, and the ratio had typical values within the optically thin range. The intensity of individual Fe II Bowen-excited lines is correlated with the C IV 1548.2 A flux, suggesting the presence of a foreground Fe II region in which fluorescent-excited material responds to flux variations of C IV 1548.2 A. In RX Pup the combined fluxes of Fe II Bowen-pumped lines can account for an appreciable fraction of the flux deficit in the C IV 1548.2 A line when the C IV doublet ratio is less than the optically thick limit of unity. The Fe II Bowen lines in RX Pup exhibit a velocity range from 0 to 80 km/s, where several strong Fe II emission lines correspond to deep absorption structure in the C IV 1548.2 A line profile. In AG Peg and C IV 1548.2 A flux deficit cannot be explained by Fe II fluorescent absorption alone when the C IV doublet ratio anomaly is at an extreme.

Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells

PubMed Central

Lubbeck, Jennifer L.; Dean, Kevin M.; Ma, Hairong; Palmer, Amy E.; Jimenez, Ralph

2012-01-01

Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts, however the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, non-temporally-coincident excitation beams. Here, we characterize the design and operation of a cytometer with a 3-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW – 179 kW/cm2). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: Sbeam3/Sbeam1) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multi-channel fluorescence cytometry, and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching. PMID:22424298

Segmentation-based retrospective shading correction in fluorescence microscopy E. coli images for quantitative analysis

NASA Astrophysics Data System (ADS)

Mai, Fei; Chang, Chunqi; Liu, Wenqing; Xu, Weichao; Hung, Yeung S.

2009-10-01

Due to the inherent imperfections in the imaging process, fluorescence microscopy images often suffer from spurious intensity variations, which is usually referred to as intensity inhomogeneity, intensity non uniformity, shading or bias field. In this paper, a retrospective shading correction method for fluorescence microscopy Escherichia coli (E. Coli) images is proposed based on segmentation result. Segmentation and shading correction are coupled together, so we iteratively correct the shading effects based on segmentation result and refine the segmentation by segmenting the image after shading correction. A fluorescence microscopy E. Coli image can be segmented (based on its intensity value) into two classes: the background and the cells, where the intensity variation within each class is close to zero if there is no shading. Therefore, we make use of this characteristics to correct the shading in each iteration. Shading is mathematically modeled as a multiplicative component and an additive noise component. The additive component is removed by a denoising process, and the multiplicative component is estimated using a fast algorithm to minimize the intra-class intensity variation. We tested our method on synthetic images and real fluorescence E.coli images. It works well not only for visual inspection, but also for numerical evaluation. Our proposed method should be useful for further quantitative analysis especially for protein expression value comparison.

Intensity ratio to improve black hole assessment in multiple sclerosis.

PubMed

Adusumilli, Gautam; Trinkaus, Kathryn; Sun, Peng; Lancia, Samantha; Viox, Jeffrey D; Wen, Jie; Naismith, Robert T; Cross, Anne H

2018-01-01

Improved imaging methods are critical to assess neurodegeneration and remyelination in multiple sclerosis. Chronic hypointensities observed on T1-weighted brain MRI, "persistent black holes," reflect severe focal tissue damage. Present measures consist of determining persistent black holes numbers and volumes, but do not quantitate severity of individual lesions. Develop a method to differentiate black and gray holes and estimate the severity of individual multiple sclerosis lesions using standard magnetic resonance imaging. 38 multiple sclerosis patients contributed images. Intensities of lesions on T1-weighted scans were assessed relative to cerebrospinal fluid intensity using commercial software. Magnetization transfer imaging, diffusion tensor imaging and clinical testing were performed to assess associations with T1w intensity-based measures. Intensity-based assessments of T1w hypointensities were reproducible and achieved > 90% concordance with expert rater determinations of "black" and "gray" holes. Intensity ratio values correlated with magnetization transfer ratios (R = 0.473) and diffusion tensor imaging metrics (R values ranging from 0.283 to -0.531) that have been associated with demyelination and axon loss. Intensity ratio values incorporated into T1w hypointensity volumes correlated with clinical measures of cognition. This method of determining the degree of hypointensity within multiple sclerosis lesions can add information to conventional imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Diffusion behavior of the fluorescent proteins eGFP and Dreiklang in solvents of different viscosity monitored by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

2016-12-01

Fluorescence correlation spectroscopy relies on temporal autocorrelation analysis of fluorescence intensity fluctuations that spontaneously arise in systems at equilibrium due to molecular motion and changes of state that cause changes in fluorescence, such as triplet state transition, photoisomerization and other photophysical transformations, to determine the rates of these processes. The stability of a fluorescent molecule against dark state conversion is of particular concern for chromophores intended to be used as reference tags for comparing diffusion processes on multiple time scales. In this work, we analyzed properties of two fluorescent proteins, the photoswitchable Dreiklang and its parental eGFP, in solvents of different viscosity to vary the diffusion time through the observation volume element by several orders of magnitude. In contrast to eGFP, Dreiklang undergoes a dark-state conversion on the time scale of tens to hundreds of microseconds under conditions of intense fluorescence excitation, which results in artificially shortened diffusion times if the diffusional motion through the observation volume is sufficiently slowed down. Such photophysical quenching processes have also been observed in FCS studies on other photoswitchable fluorescent proteins including Citrine, from which Dreiklang was derived by genetic engineering. This property readily explains the discrepancies observed previously between the diffusion times of eGFP- and Dreiklang-labeled plasma membrane protein complexes.

Fluorescent optical position sensor

DOEpatents

Weiss, Jonathan D.

2005-11-15

A fluorescent optical position sensor and method of operation. A small excitation source side-pumps a localized region of fluorescence at an unknown position along a fluorescent waveguide. As the fluorescent light travels down the waveguide, the intensity of fluorescent light decreases due to absorption. By measuring with one (or two) photodetectors the attenuated intensity of fluorescent light emitted from one (or both) ends of the waveguide, the position of the excitation source relative to the waveguide can be determined by comparing the measured light intensity to a calibrated response curve or mathematical model. Alternatively, excitation light can be pumped into an end of the waveguide, which generates an exponentially-decaying continuous source of fluorescent light along the length of the waveguide. The position of a photodetector oriented to view the side of the waveguide can be uniquely determined by measuring the intensity of the fluorescent light emitted radially at that location.

[Combined application of multiple fluorescence in research on the degradation of fluoranthene by potassium ferrate].

PubMed

Li, Si; Yu, Dan-Ni; Ji, Fang-Ying; Zhou, Guang-Ming; He, Qiang

2012-11-01

The degradation of fluoranthene was researched by combined means of multiple fluorescence spectra, including emission, synchronous, excitation emission matrix (EEM), time-scan and photometry. The characteristics of the degradation and fluoranthene molecular changes within the degradation's process were also discussed according to the information about the degradation provided by all of the fluorescence spectra mentioned above. The equations of fluoranthene's degradation by potassium ferrate were obtained on the bases of fitting time-scan fluorescence curves at different time, and the degradation's kinetic was speculated accordingly. From the experimental results, multiple fluorescence data commonly reflected that it had same degradation rate at the same reaction time. t = 10 s, and the degradation rate is -55%, t = 25 s, -81%, t = 40 s, -91%. No new fluorescent characteristic was observed within every degradation' stage. The reaction stage during t < or = 20 s was crucial, in which the degradation process is closest to linear relationship. After this beginning stage, the linear relationship deviated gradually with the development of the degradation process. The degradation of fluoranthene by potassium ferrate was nearly in accord with the order of the first order reaction.

Comparative study of the fluorescence intensity of dental composites and human teeth submitted to artificial aging.

PubMed

Jablonski, Tatiana; Takahashi, Marcos Kenzo; Brum, Rafeal Torres; Rached, Rodrigo Nunes; Souza, Evelise M

2014-01-01

The aim of this study was to evaluate quantitatively the fluorescence of resin composites and human teeth, and to determine the stability of fluorescence after aging. Ten specimens were built using a 1 mm thick increment of dentin composite overlapped by a 0.5 mm thick increment of enamel composite. Ten sound human molars were sectioned and silicon carbide-polished to obtain enamel and dentin slabs 1.5 mm in thickness. Fluorescence measurements were carried out by a fluorescence spectrophotometer before and after thermocycling (2000 cycles, 5°C and 55°C). One-way analysis of variance (ANOVA) with repeated measures and Tukey's test were performed at a significance level of 5%. Most of the tested composites showed significant differences in fluorescence both before and after aging (P < 0.05). Opallis was the only composite whose fluorescence was similar to that of human teeth at both periods of evaluation (P > 0.05), and was the only composite that showed comparable results of fluorescence to the tooth structure before and after thermocycling. With the exception of Filtek Supreme, there were significant reductions in fluorescence intensity for all the tested composites (P < 0.05).

Plasmonic platforms of self-assembled silver nanostructures in application to fluorescence

PubMed Central

Luchowski, Rafal; Calander, Nils; Shtoyko, Tanya; Apicella, Elisa; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

2011-01-01

Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2–3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from localized areas of silver nanostructures. PMID:21403765

Fluorescent fiber diagnostics

DOEpatents

Toeppen, John S.

1994-10-04

A fluorescent fiber (13) having a doped core (16) is pumped (11) by light (18) of a relatively short wavelength to produce fluorescence at a longer wavelength that is detected by detector (24). The level of fluorescence is monitored (26) and evaluated to provide information as to the excitation of the fiber (13) or the environment thereof. In particular, the level of intensity of the detected fluorescence may be used to measure the intensity of a light beam (18) passing axially through an optical fiber system (12) (FIG. 1 ), or the intensity of a light beam (46) passing radially through a fluorescent fiber (13) (FIG. 2 ), or the level of a fluid (32) in a tank (31) (FIG. 3 ), or a scintillation event (37) in a fluorescent fiber (13) pumped to produce amplification of the scintillation event (FIG. 4 ).

Fluorescent fiber diagnostics

DOEpatents

Toeppen, John S.

1994-01-01

A fluorescent fiber (13) having a doped core (16) is pumped (11) by light (18) of a relatively short wavelength to produce fluorescence at a longer wavelength that is detected by detector (24). The level of fluorescence is monitored (26) and evaluated to provide information as to the excitation of the fiber (13) or the environment thereof. In particular, the level of intensity of the detected fluorescence may be used to measure the intensity of a light beam (18) passing axially through an optical fiber system (12) (FIG. 1 ), or the intensity of a light beam (46) passing radially through a fluorescent fiber (13) (FIG. 2 ), or the level of a fluid (32) in a tank (31) (FIG. 3 ), or a scintillation event (37) in a fluorescent fiber (13) pumped to produce amplification of the scintillation event (FIG. 4 ).

Aspect ratio dependence of the enhancement of fluorescence intensity by gold nanobipyramids for cancer cell imaging and photodynamic therapy

NASA Astrophysics Data System (ADS)

Wang, Jing; Wang, Shimiao; Mi, Lan; Liu, Jun

2018-07-01

Enhancement of dye fluorescence intensity was studied by modifying the aspect ratio of gold nanobipyramids (AuBPs) from 3.2 to 6.6. The emission fluorescence intensity of sulfonated aluminum phthalocyanine (AlPcS) was strongly dependent on the aspect ratio of AuBPs. Furthermore, we found that the energy transfer from excited AlPcS to AuBPs was a key determinant of the efficacy of metal-enhanced fluorescence. By means of AuBPs with a higher aspect ratio, such that the surface plasmon resonance band does not overlap with the energy level of excited AlPcS, metal-enhanced fluorescence of various AlPcS–AuBP conjugates was determined, and the maximal enhancement factor was found to be 14. The enhanced fluorescence intensity of AlPcS conjugated with AuBPs indicates promising plasmonic properties. An apoptosis assay of HeLa cells revealed that AlPcS–AuBPs, when used as a drug, can enhance the effectiveness of photodynamic therapy (PDT). Furthermore, AuBPs with the longitudinal absorption peak wavelength of 1050 nm had optimal proapoptotic effects. HeLa cells treated with AlPcS–AuBPs (ratio 0.42 µM to 0.01 nM) had viability as low as 29.31% after 32 J cm‑2 ultraviolet light exposure, indicating the strong potential of AlPcS–AuBPs to improve the efficacy of PDT.

Confocal microscopy evaluation of stromal fluorescence intensity after standard and accelerated iontophoresis-assisted corneal cross-linking.

PubMed

Lanzini, Manuela; Curcio, Claudia; Spoerl, Eberhard; Calienno, Roberta; Mastropasqua, Alessandra; Colasante, Martina; Mastropasqua, Rodolfo; Nubile, Mario; Mastropasqua, Leonardo

2017-02-01

The aim of this study is to determine modifications in stromal fluorescence intensity after different corneal cross-linking (CXL) procedures and to correlate stromal fluorescence to corneal biomechanical resistance. For confocal microscopy study, 15 human cadaver corneas were examined. Three served as control (group 1), three were just soaked with iontophoresis procedure (group 2), three were treated with standard epi-off technique (group 3), and six underwent iontophoresis imbibition. Three of later six were irradiated for 30 min with 3 mW/cm 2 UVA (group 4) and three for 9 min at 10 mW/cm 2 UVA (group 5). Confocal microscopy was performed to quantify the fluorescence intensity in the cornea at different stromal depths. For biomechanical study, 30 human cadaver corneas were randomly divided into five groups and treated as previously described. Static stress-strain measurements of the corneas were performed. Iontophoresis imbibition followed by 10mW/cm 2 irradiation proved to increase stromal fluorescence into the corneal stroma and significant differences were revealed between group 3 and 5 both at 100 (p = 0.0171) and 250 µm (p = 0.0024), respectively. Biomechanical analysis showed an improvement of corneal resistance in group 5. Iontophoresis imbibition followed by accelerated irradiation increased the stromal fluorescence and is related to an improvement of biomechanical resistance. This approach may represent a new strategy to achieve greater concentrations of riboflavin without removing corneal epithelium and improve clinical results while reducing the side effects of CXL.

Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

PubMed Central

Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

2016-01-01

We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

Handheld Fluorescence Microscopy based Flow Analyzer.

PubMed

Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

2016-03-01

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images.

PubMed

Ferro, Anabela; Mestre, Tânia; Carneiro, Patrícia; Sahumbaiev, Ivan; Seruca, Raquel; Sanches, João M

2017-05-01

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

PubMed Central

Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

2012-01-01

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty

Effects of water stress and light intensity on chlorophyll fluorescence parameters and pigments of Aloe vera L.

PubMed

Hazrati, Saeid; Tahmasebi-Sarvestani, Zeinolabedin; Modarres-Sanavy, Seyed Ali Mohammad; Mokhtassi-Bidgoli, Ali; Nicola, Silvana

2016-09-01

Aloe vera L. is one of the most important medicinal plants in the world. In order to determine the effects of light intensity and water deficit stress on chlorophyll (Chl) fluorescence and pigments of A. vera, a split-plot in time experiment was laid out in a randomized complete block design with four replications in a research greenhouse. The factorial combination of three light intensities (50, 75 and 100% of sunlight) and four irrigation regimes (irrigation after depleting 20, 40, 60 and 80% of soil water content) were considered as main factors. Sampling time was considered as sub factor. The first, second and third samplings were performed 90, 180 and 270 days after imposing the treatments, respectively. The results demonstrated that the highest light intensity and the severe water stress decreased maximum fluorescence (Fm), variable fluorescence (Fv)/Fm, quantum yield of PSII photochemistry (ФPSII), Chl and photochemical quenching (qP) but increased non-photochemical quenching (NPQ), minimum fluorescence (F0) and Anthocyanin (Anth). Additionally, the highest Fm, Fv/Fm, ФPSII and qP and the lowest NPQ and F0 were observed when 50% of sunlight was blocked and irrigation was done after 40% soil water depletion. Irradiance of full sunlight and water deficit stress let to the photoinhibition of photosynthesis, as indicated by a reduced quantum yield of PSII, ФPSII, and qP, as well as higher NPQ. Thus, chlorophyll florescence measurements provide valuable physiological data. Close to half of total solar radiation and irrigation after depleting 40% of soil water content were selected as the most efficient treatments. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Using water raman intensity to determine the effective excitation and emission path lengths of fluorophotometers for correcting fluorescence inner filter effect

USDA-ARS?s Scientific Manuscript database

Fluorescence and Raman inner filter effects (IFE) cause spectral distortion and nonlinearity between spectral signal intensity with increasing analyte concentration. Convenient and effective correction of fluorescence IFE has been an active research goal for decades. Presented herein is the finding ...

Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

PubMed

Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

2009-01-01

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

In vivo optical imaging of dihydroethidium oxidation in the mouse brain employing fluorescence intensity and lifetime contrast

NASA Astrophysics Data System (ADS)

Hall, David J.; Han, Sung-Ho; Dugan, Laura

2009-02-01

Reactive oxygen species (ROS) are believed to be involved in many diseases and injuries to the brain, but the molecular processes are not well understood due to a lack of in vivo imaging techniques to evaluate ROS. The fluorescent oxidation products of dihydroethidium (DHE) can monitor ROS production in vivo. Here we demonstrate the novel optical imaging of brain in live mice to measure ROS production via generation of fluorescent DHE oxidation products (ox-DHE) by ROS. We show that in Sod2+/- mice, which have partial loss of a key antioxidant enzyme, superoxide dismutase-2, that ox-DHE fluorescence intensity was significantly higher than in hSOD1 mice, which have four-fold overexpression of superoxide dismutase-1 activity, which had almost no ox-DHE fluorescence, confirming specificity of ox-DHE to ROS production. The DHE oxidation products were also confirmed by detecting a characteristic fluorescence lifetime of the oxidation product, which was validated with ex vivo measurements.

A Solar-Pumped Fluorescence Model for Line-By-Line Emission Intensities in the B-X, A-X, and X-X Band Systems of 12C14N

NASA Technical Reports Server (NTRS)

Paganini, L.; Mumma, M. J.

2016-01-01

We present a new quantitative model for detailed solar-pumped fluorescent emission of the main isotopologue of CN. The derived fluorescence efficiencies permit estimation and interpretation of ro-vibrational infrared line intensities of CN in exospheres exposed to solar (or stellar) radiation. Our g-factors are applicable to astronomical observations of CN extending from infrared to optical wavelengths, and we compare them with previous calculations in the literature. The new model enables extraction of rotational temperature, column abundance, and production rate from astronomical observations of CN in the inner coma of comets. Our model accounts for excitation and de-excitation of rotational levels in the ground vibrational state by collisions, solar excitation to the A(sup 2)Pi(sub I) and B(sup 2)Sum(sup +) electronically excited states followed by cascade to ro-vibrational levels of X(sup 2)Sum(sup +), and direct solar infrared pumping of ro-vibrational levels in the X(sup 2)Sum(sup +) state. The model uses advanced solar spectra acquired at high spectral resolution at the relevant infrared and optical wavelengths and considers the heliocentric radial velocity of the comet (the Swings effect) when assessing the exciting solar flux for a given transition. We present model predictions for the variation of fluorescence rates with rotational temperature and heliocentric radial velocity. Furthermore, we test our fluorescence model by comparing predicted and measured line-by-line intensities for X(sup 2)Sum(sup +) (1-0) in comet C/2014 Q2 (Lovejoy), thereby identifying multiple emission lines observed at IR wavelengths.

Polar plot representation of time-resolved fluorescence.

PubMed

Eichorst, John Paul; Wen Teng, Kai; Clegg, Robert M

2014-01-01

Measuring changes in a molecule's fluorescence emission is a common technique to study complex biological systems such as cells and tissues. Although the steady-state fluorescence intensity is frequently used, measuring the average amount of time that a molecule spends in the excited state (the fluorescence lifetime) reveals more detailed information about its local environment. The lifetime is measured in the time domain by detecting directly the decay of fluorescence following excitation by short pulse of light. The lifetime can also be measured in the frequency domain by recording the phase and amplitude of oscillation in the emitted fluorescence of the sample in response to repetitively modulated excitation light. In either the time or frequency domain, the analysis of data to extract lifetimes can be computationally intensive. For example, a variety of iterative fitting algorithms already exist to determine lifetimes from samples that contain multiple fluorescing species. However, recently a method of analysis referred to as the polar plot (or phasor plot) is a graphical tool that projects the time-dependent features of the sample's fluorescence in either the time or frequency domain into the Cartesian plane to characterize the sample's lifetime. The coordinate transformations of the polar plot require only the raw data, and hence, there are no uncertainties from extensive corrections or time-consuming fitting in this analysis. In this chapter, the history and mathematical background of the polar plot will be presented along with examples that highlight how it can be used in both cuvette-based and imaging applications.

Tracking multiple particles in fluorescence time-lapse microscopy images via probabilistic data association.

PubMed

Godinez, William J; Rohr, Karl

2015-02-01

Tracking subcellular structures as well as viral structures displayed as 'particles' in fluorescence microscopy images yields quantitative information on the underlying dynamical processes. We have developed an approach for tracking multiple fluorescent particles based on probabilistic data association. The approach combines a localization scheme that uses a bottom-up strategy based on the spot-enhancing filter as well as a top-down strategy based on an ellipsoidal sampling scheme that uses the Gaussian probability distributions computed by a Kalman filter. The localization scheme yields multiple measurements that are incorporated into the Kalman filter via a combined innovation, where the association probabilities are interpreted as weights calculated using an image likelihood. To track objects in close proximity, we compute the support of each image position relative to the neighboring objects of a tracked object and use this support to recalculate the weights. To cope with multiple motion models, we integrated the interacting multiple model algorithm. The approach has been successfully applied to synthetic 2-D and 3-D images as well as to real 2-D and 3-D microscopy images, and the performance has been quantified. In addition, the approach was successfully applied to the 2-D and 3-D image data of the recent Particle Tracking Challenge at the IEEE International Symposium on Biomedical Imaging (ISBI) 2012.

Intensity and amplitude correlations in the fluorescence from atoms with interacting Rydberg states

NASA Astrophysics Data System (ADS)

Xu, Qing; Mølmer, Klaus

2015-09-01

We explore the fluorescence signals from a pair of atoms driven towards Rydberg states on a three-level ladder transition. The dipole-dipole interactions between Rydberg excited atoms significantly distort the dark state and electromagnetically induced transparency behavior observed with independent atoms and, thus, their steady-state light emission. We calculate and analyze the temporal correlations between intensities and amplitudes of the signals emitted by the atoms and explain their origin in the atomic Rydberg state interactions.

Methods for Broadband Spectral Analysis: Intrinsic Fluorescence Temperature Sensing as an Example.

PubMed

Zhang, Weiwei; Wang, Guoyao; Baxter, Greg W; Collins, Stephen F

2017-06-01

A systematic study was performed on the temperature-dependent fluorescence of (Ba,Sr) 2 SiO 4 :Eu 2+ . The barycenter and extended intensity ratio techniques were proposed to characterize the broadband fluorescence spectra. These techniques and other known methods (listed below) were employed and compared in the fluorescent temperature sensing experiment. Multiple sensing functions were obtained using the behaviors of: (1) the barycenter location of the emission band; (2) the emission bandwidth; and (3) the ratio of intensities at different wavelengths in the emission band, respectively. The barycenter technique was not limited by the spectrometer resolution and worked well while the peak location method failed. All the sensing functions were based on the intrinsic characteristics of the fluorescence of the phosphor and demonstrated nearly linear relationships with temperature in the measuring range. The multifunctional temperature-sensing abilities of the phosphor can be applied in a point thermometer or thermal mapping. The new techniques were validated successfully for characterizing various spectra.

Chromosome characterization using single fluorescent dye

DOEpatents

Crissman, Harry A.; Hirons, Gregory T.

1995-01-01

Chromosomes are characterized by fluorescent emissions from a single fluorescent dye that is excited over two different wavelengths. A mixture containing chromosomes is stained with a single dye selected from the group consisting of TOTO and YOYO and the stained chromosomes are placed in a flow cytometer. The fluorescent dye is excited sequentially by a first light having a wavelength in the ultraviolet range to excite the TOTO or YOYO to fluoresce at a first intensity and by a second light having a wavelength effective to excite the TOTO or YOYO dye to fluoresce at a second intensity. Specific chromosomes may be identified and sorted by intensity relationships between the first and second fluorescence emissions.

Multiple signal classification algorithm for super-resolution fluorescence microscopy

PubMed Central

Agarwal, Krishna; Macháň, Radek

2016-01-01

Single-molecule localization techniques are restricted by long acquisition and computational times, or the need of special fluorophores or biologically toxic photochemical environments. Here we propose a statistical super-resolution technique of wide-field fluorescence microscopy we call the multiple signal classification algorithm which has several advantages. It provides resolution down to at least 50 nm, requires fewer frames and lower excitation power and works even at high fluorophore concentrations. Further, it works with any fluorophore that exhibits blinking on the timescale of the recording. The multiple signal classification algorithm shows comparable or better performance in comparison with single-molecule localization techniques and four contemporary statistical super-resolution methods for experiments of in vitro actin filaments and other independently acquired experimental data sets. We also demonstrate super-resolution at timescales of 245 ms (using 49 frames acquired at 200 frames per second) in samples of live-cell microtubules and live-cell actin filaments imaged without imaging buffers. PMID:27934858

Fluorescence Behavior and Dural Infiltration of Meningioma Analyzed by 5-Aminolevulinic Acid-Based Fluorescence: Operating Microscope Versus Mini-Spectrometer.

PubMed

Knipps, Johannes; Beseoglu, Kerim; Kamp, Marcel; Fischer, Igor; Felsberg, Joerg; Neumann, Lisa M; Steiger, Hans-Jakob; Cornelius, Jan F

2017-12-01

To compare fluorescence intensity of tumor specimens, as measured by a fluorescence-guided surgery microscope and a spectrometer, to evaluate tumor infiltration of dura mater around meningiomas with help of these 2 different 5-aminolevulinic acid (5-ALA)-based fluorescence tools, and to correlate fluorescence intensity with histopathologic data. In a clinical series, meningiomas were resected by 5-ALA fluorescence-guided surgery. Fluorescence intensity was semiquantitatively rated by the surgeon at predefined points. Biopsies were harvested and fluorescence intensity measured by a spectrometer and histopathologically analyzed. Sampling was realized at the level of the dura in a centrifugal direction. A total of 104 biopsies (n = 13 tumors) were analyzed. Specificity and sensitivity of the microscope were 0.96 and 0.53 and of the spectrometer 0.95 and 0.93, respectively. Fluorescence intensity as measured by the spectrometer was correlated to histologically confirmed tumor burden. In a centrifugal direction, tumor burden and fluorescence intensity continuously decreased (along the dural tail). Below a threshold value of 639 arbitrary units no tumor was histologically detectable. At the level of the dura the spectrometer was highly sensitive for detection of meningioma cells. The surgical microscope showed false negative results and missed residual tumor cells in more than one half of the cases. The complementary use of both fluorescence tools may improve resection quality. Copyright © 2017 Elsevier Inc. All rights reserved.

Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

2016-03-01

Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

PubMed Central

Petersen, Karl J.; Peterson, Kurt C.; Muretta, Joseph M.; Higgins, Sutton E.; Gillispie, Gregory D.; Thomas, David D.

2014-01-01

We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5–10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications. PMID:25430092

Accurate thermometry based on the red and green fluorescence intensity ratio in NaYF₄: Yb, Er nanocrystals for bioapplication.

PubMed

Liu, Lixin; Qin, Feng; Lv, Tianquan; Zhang, Zhiguo; Cao, Wenwu

2016-10-15

A biological temperature measurement method based on the fluorescence intensity ratio (FIR) was developed to reduce uncertainty. The upconversion luminescence of NaYF₄:Yb, Er nanocrystals was studied as a function of temperature around the physiologically relevant range of 300-330 K. We found that the green-green FIR Fe and red-green FIR (I₆₆₀/I₅₄₀) varied linearly as temperature increased. The thermometric uncertainties using the two FIRs were discussed and were determined to be almost constant at 0.6 and 0.09 K for green-green and red-green, respectively. The lower thermometric uncertainty comes from the intense signal-to-noise ratio of the measured FIRs owing to their comparable fluorescence intensities.

Self-interference fluorescence microscopy with three-phase detection for depth-resolved confocal epi-fluorescence imaging.

PubMed

Braaf, Boy; de Boer, Johannes F

2017-03-20

Three-dimensional confocal fluorescence imaging of in vivo tissues is challenging due to sample motion and limited imaging speeds. In this paper a novel method is therefore presented for scanning confocal epi-fluorescence microscopy with instantaneous depth-sensing based on self-interference fluorescence microscopy (SIFM). A tabletop epi-fluorescence SIFM setup was constructed with an annular phase plate in the emission path to create a spectral self-interference signal that is phase-dependent on the axial position of a fluorescent sample. A Mach-Zehnder interferometer based on a 3 × 3 fiber-coupler was developed for a sensitive phase analysis of the SIFM signal with three photon-counter detectors instead of a spectrometer. The Mach-Zehnder interferometer created three intensity signals that alternately oscillated as a function of the SIFM spectral phase and therefore encoded directly for the axial sample position. Controlled axial translation of fluorescent microsphere layers showed a linear dependence of the SIFM spectral phase with sample depth over axial image ranges of 500 µm and 80 µm (3.9 × Rayleigh range) for 4 × and 10 × microscope objectives respectively. In addition, SIFM was in good agreement with optical coherence tomography depth measurements on a sample with indocyanine green dye filled capillaries placed at multiple depths. High-resolution SIFM imaging applications are demonstrated for fluorescence angiography on a dye-filled capillary blood vessel phantom and for autofluorescence imaging on an ex vivo fly eye.

Potential rainfall-intensity and pH-driven shifts in the apparent fluorescent composition of dissolved organic matter in rainwater.

PubMed

Zhou, Yongqiang; Yao, Xiaolong; Zhang, Yibo; Shi, Kun; Zhang, Yunlin; Jeppesen, Erik; Gao, Guang; Zhu, Guangwei; Qin, Boqiang

2017-05-01

Perturbations of rainwater chromophoric dissolved organic matter (CDOM) fluorescence induced by changes in rainfall intensity and pH were investigated by field observations and laboratory pH titrations. Microbial humic-like fluorophores dominated the rainwater CDOM pool, followed by tryptophan-like and tyrosine-like substances. Increased rainfall intensity had notable dilution effects on all six fluorescent components (C1-C6) identified using parallel factor (PARAFAC) analysis, the effect being especially pronounced for the microbial humic-like C1, tryptophan-like C3, and tyrosine-like C5. The results also indicated that increasing pH from 7 to 9 led to decreased fluorescence intensity (F max ) of all the six components, while a pH increase from 5 to 7, resulted in increasing F max of terrestrial humic-like C2, tyrosine-like C5, and tryptophan-like C6 and decreasing microbial humic-like C1, tryptophan-like C3, and fulvic-like C4. Two-dimensional correlation spectroscopy (2D-COS) demonstrated that synchronous fluorescence responded first to pH modifications at fulvic-like wavelength (λ Ex /λ Em  = ∼316/416 nm), followed by tyrosine-like wavelength (λ Ex /λ Em  = ∼204/304 nm), tryptophan-like wavelength (λ Ex /λ Em  = ∼226/326 nm), microbial humic-like wavelength (∼295/395 nm), and finally terrestrial humic-like wavelength (∼360/460 nm). Our results suggest that a decrease in areas affected by acid rain in South China occurring at present may possibly result in apparent compositional changes of CDOM fluorescence. The decreased rainfall in South-West China and increased rainfall in North-West China during the past five decades may possibly accordingly result in increased and decreased F max of all the six components identified in South-West and North-West China, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ex-vivo UV autofluorescence imaging and fluorescence spectroscopy of atherosclerotic pathology in human aorta

NASA Astrophysics Data System (ADS)

Lewis, William; Williams, Maura; Franco, Walfre

2017-02-01

The aim of our study was to identify fluorescence excitation-emission pairs correlated with atherosclerotic pathology in ex-vivo human aorta. Wide-field images of atherosclerotic human aorta were captured using UV and visible excitation and emission wavelength pairs of several known fluorophores to investigate correspondence with gross pathologic features. Fluorescence spectroscopy and histology were performed on 21 aortic samples. A matrix of Pearson correlation coefficients were determined for the relationship between relevant histologic features and the intensity of emission for 427 wavelength pairs. A multiple linear regression analysis indicated that elastin (370/460 nm) and tryptophan (290/340 nm) fluorescence predicted 58% of the variance in intima thickness (R-squared = 0.588, F(2,18) = 12.8, p=.0003), and 48% of the variance in media thickness (R-squared = 0.483, F(2,18) = 8.42, p=.002), suggesting that endogenous fluorescence intensity at these wavelengths can be utilized for improved pathologic characterization of atherosclerotic plaques.

Fluorescent probes for the simultaneous detection of multiple analytes in biology.

PubMed

Kolanowski, Jacek L; Liu, Fei; New, Elizabeth J

2018-01-02

Many of the key questions facing cellular biology concern the location and concentration of chemical species, from signalling molecules to metabolites to exogenous toxins. Fluorescent sensors (probes) have revolutionised the understanding of biological systems through their exquisite sensitivity to specific analytes. Probe design has focussed on selective sensors for individual analytes, but many of the most pertinent biological questions are related to the interaction of more than one chemical species. While it is possible to simultaneously use multiple sensors for such applications, data interpretation will be confounded by the fact that sensors will have different uptake, localisation and metabolism profiles. An alternative solution is to instead use a single probe that responds to two analytes, termed a dual-responsive probe. Recent progress in this field has yielded exciting probes, some of which have demonstrated biological application. Here we review work that has been carried out to date, and suggest future research directions that will harness the considerable potential of dual-responsive fluorescent probes.

CdSe/ZnS quantum dot fluorescence spectra shape-based thermometry via neural network reconstruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Munro, Troy; Laboratory of Soft Matter and Biophysics, Department of Physics and Astronomy, KU Leuven, Celestijnenlaan 200D, B-3001 Heverlee; Liu, Liwang

As a system of interest gets small, due to the influence of the sensor mass and heat leaks through the sensor contacts, thermal characterization by means of contact temperature measurements becomes cumbersome. Non-contact temperature measurement offers a suitable alternative, provided a reliable relationship between the temperature and the detected signal is available. In this work, exploiting the temperature dependence of their fluorescence spectrum, the use of quantum dots as thermomarkers on the surface of a fiber of interest is demonstrated. The performance is assessed of a series of neural networks that use different spectral shape characteristics as inputs (peak-based—peak intensity,more » peak wavelength; shape-based—integrated intensity, their ratio, full-width half maximum, peak normalized intensity at certain wavelengths, and summation of intensity over several spectral bands) and that yield at their output the fiber temperature in the optically probed area on a spider silk fiber. Starting from neural networks trained on fluorescence spectra acquired in steady state temperature conditions, numerical simulations are performed to assess the quality of the reconstruction of dynamical temperature changes that are photothermally induced by illuminating the fiber with periodically intensity-modulated light. Comparison of the five neural networks investigated to multiple types of curve fits showed that using neural networks trained on a combination of the spectral characteristics improves the accuracy over use of a single independent input, with the greatest accuracy observed for inputs that included both intensity-based measurements (peak intensity) and shape-based measurements (normalized intensity at multiple wavelengths), with an ultimate accuracy of 0.29 K via numerical simulation based on experimental observations. The implications are that quantum dots can be used as a more stable and accurate fluorescence thermometer for solid materials and that

Diurnal Variability in Chlorophyll-a, Carotenoids, CDOM and SO₄(2-) Intensity of Offshore Seawater Detected by an Underwater Fluorescence-Raman Spectral System.

PubMed

Chen, Jing; Ye, Wangquan; Guo, Jinjia; Luo, Zhao; Li, Ying

2016-07-13

A newly developed integrated fluorescence-Raman spectral system (λex = 532 nm) for detecting Chlorophyll-a (chl-a), Chromophoric Dissolved Organic Matter (CDOM), carotenoids and SO₄(2-) in situ was used to successfully investigate the diurnal variability of all above. Simultaneously using the integration of fluorescence spectroscopy and Raman spectroscopy techniques provided comprehensive marine information due to the complementarity between the different excitation mechanisms and different selection rules. The investigation took place in offshore seawater of the Yellow Sea (36°05'40'' N, 120°31'32'' E) in October 2014. To detect chl-a, CDOM, carotenoids and SO₄(2-), the fluorescence-Raman spectral system was deployed. It was found that troughs of chl-a and CDOM fluorescence signal intensity were observed during high tides, while the signal intensity showed high values with larger fluctuations during ebb-tide. Chl-a and carotenoids were influenced by solar radiation within a day cycle by different detection techniques, as well as displaying similar and synchronous tendency. CDOM fluorescence cause interference to the measurement of SO₄(2-). To avoid such interference, the backup Raman spectroscopy system with λex = 785 nm was employed to detect SO₄(2-) concentration on the following day. The results demonstrated that the fluorescence-Raman spectral system has great potential in detection of chl-a, carotenoids, CDOM and SO₄(2-) in the ocean.

The Intensity Modulation of the Fluorescent Line by a Finite Light Speed Effect in Accretion-powered X-Ray Pulsars

NASA Astrophysics Data System (ADS)

Yoshida, Yuki; Kitamoto, Shunji; Hoshino, Akio

2017-11-01

The X-ray line diagnostic method is a powerful tool for an investigation of plasma around accretion-powered X-ray pulsars. We point out an apparent intensity modulation of emission lines, with their rotation period of neutron stars, due to the finite speed of light (we call this effect the “finite light speed effect”) if the line emission mechanism is a kind of reprocessing, such as fluorescence or recombination after ionization by X-ray irradiation from pulsars. The modulation amplitude is determined by the size of the emission region, which is in competition with the smearing effect by the light crossing time in the emission region. This is efficient if the size of the emission region is roughly comparable to that of the rotation period multiplied by the speed of light. We apply this effect to a symbiotic X-ray pulsar, GX 1+4, where a spin modulation of the intense iron line of which has been reported. The finite light speed effect can explain the observed intensity modulation if its fluorescent region is the size of ˜ {10}12 cm.

Multicolor fluorescence enhancement from a photonics crystal surface

NASA Astrophysics Data System (ADS)

Pokhriyal, A.; Lu, M.; Huang, C. S.; Schulz, S.; Cunningham, B. T.

2010-09-01

A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, fabricated uniformly from plastic materials over a ˜3×5 in.2 surface area by nanoreplica molding, utilizes two distinct resonant modes to enhance electric field stimulation of a dye excited by a λ =632.8 nm laser (cyanine-5) and a dye excited by a λ =532 nm laser (cyanine-3). Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle. Compared to detection of a dye-labeled protein on an ordinary glass surface, the photonic crystal surface exhibited a 32× increase in fluorescent signal intensity for cyanine-5 conjugated streptavidin labeling, while a 25× increase was obtained for cyanine-3 conjugated streptavidin labeling. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in the 532 nm<λ<633 nm range by selection of an appropriate incident angle. The device is designed for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays.

Multicolor fluorescence enhancement from a photonics crystal surface

PubMed Central

Pokhriyal, A.; Lu, M.; Huang, C. S.; Schulz, S.; Cunningham, B. T.

2010-01-01

A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, fabricated uniformly from plastic materials over a ∼3×5 in.2 surface area by nanoreplica molding, utilizes two distinct resonant modes to enhance electric field stimulation of a dye excited by a λ=632.8 nm laser (cyanine-5) and a dye excited by a λ=532 nm laser (cyanine-3). Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle. Compared to detection of a dye-labeled protein on an ordinary glass surface, the photonic crystal surface exhibited a 32× increase in fluorescent signal intensity for cyanine-5 conjugated streptavidin labeling, while a 25× increase was obtained for cyanine-3 conjugated streptavidin labeling. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in the 532 nm<λ<633 nm range by selection of an appropriate incident angle. The device is designed for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays. PMID:20957067

Correlation fluorescence method of amine detection

NASA Astrophysics Data System (ADS)

Myslitsky, Valentin F.; Tkachuk, Svetlana S.; Rudeichuk, Volodimir M.; Strinadko, Miroslav T.; Slyotov, Mikhail M.; Strinadko, Marina M.

1997-12-01

The amines fluorescence spectra stimulated by UV laser radiation are investigated in this paper. The fluorescence is stimulated by the coherent laser beam with the wavelength 0.337 micrometers . At the sufficient energy of laser stimulation the narrow peaks of the fluorescence spectra are detected besides the wide maximum. The relationship between the fluorescence intensity and the concentration of amines solutions are investigated. The fluorescence intensity temporal dependence on wavelength 0.363 micrometers of the norepinephrine solution preliminarily radiated by UV laser with wavelength 0.337 micrometers was found. The computer stimulated and experimental investigations of adrenaline and norepinephrine mixtures fluorescence spectra were done. The correlation fluorescent method of amines detection is proposed.

Photoinduced fluorescence intensity oscillation in a reaction-diffusion cell containing a colloidal quantum dot dispersion

NASA Astrophysics Data System (ADS)

Komoto, Atsushi; Maenosono, Shinya

2006-09-01

The nonlinear spontaneous oscillation of photoluminescence (PL) intensity in an ensemble of semiconductor quantum dots (QDs), which differs from the fluorescence intermittency of a single QD, is investigated. The PL intensity in a QD dispersion slowly oscillates with time under continuous illumination. The oscillatory behavior is found to vary with changing QD concentration, solvent viscosity, volume fraction of irradiated region, and irradiation intensity. On the basis of the Gray-Scott model [Chemical Oscillation and Instabilities: Non-linear Chemical Kinetics (Clarendon, Oxford, 1994); J. Phys. Chem. 89, 22 (1985); Chem. Eng. Sci. 42, 307 (1987)], and its comparison with the experimental results, it is revealed that the following processes are important for PL oscillation: (1) mass transfer of QDs between the illuminated and dark regions, (2) autocatalytic formation of vacant sites on QD surfaces via photodesorption of ligand molecules, and (3) passivation of vacant sites via photoadsorption of water molecules.

Photoinduced fluorescence intensity oscillation in a reaction-diffusion cell containing a colloidal quantum dot dispersion.

PubMed

Komoto, Atsushi; Maenosono, Shinya

2006-09-21

The nonlinear spontaneous oscillation of photoluminescence (PL) intensity in an ensemble of semiconductor quantum dots (QDs), which differs from the fluorescence intermittency of a single QD, is investigated. The PL intensity in a QD dispersion slowly oscillates with time under continuous illumination. The oscillatory behavior is found to vary with changing QD concentration, solvent viscosity, volume fraction of irradiated region, and irradiation intensity. On the basis of the Gray-Scott model [Chemical Oscillation and Instabilities: Non-linear Chemical Kinetics (Clarendon, Oxford, 1994); J. Phys. Chem. 89, 22 (1985); Chem. Eng. Sci. 42, 307 (1987)], and its comparison with the experimental results, it is revealed that the following processes are important for PL oscillation: (1) mass transfer of QDs between the illuminated and dark regions, (2) autocatalytic formation of vacant sites on QD surfaces via photodesorption of ligand molecules, and (3) passivation of vacant sites via photoadsorption of water molecules.

Real-time fluorescence microscopy monitoring of porphyrin biodistribution

NASA Astrophysics Data System (ADS)

Kimel, Sol; Gottfried, Varda; Kunzi-Rapp, Karin; Akguen, Nermin; Schneckenburger, Herbert

1996-01-01

In vivo uptake of the natural porphyrins, uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), was monitored by fluorescence microscopy. Experiments were performed using the chick chorioallantoic membrane (CAM) model, which allowed video documentation of fluorescence both in real time and after integration over a chosen time interval (usually 2 s). Sensitizers at a concentration of 50 (mu) M (100 (mu) L) were injected into a medium-sized vein (diameter approximately 40 micrometer) using an ultra-fine 10 micrometer diameter needle. Fluorescence images were quantitated by subtracting the fluorescence intensity of surrounding CAM tissue (Fmatrix) from the intravascular fluorescence intensity (Fintravascular), after transformation of the video frames into digital form. The differential fluorescence intensity, Fintravascular - Fmatrix, is a measure of the biodistribution. Real time measurements clearly showed that CP and UP fluorescence is associated with moving erythrocytes and not with endothelial cells of the vessel wall. Fluorescence intensity was monitored, up to 60 minutes after injection, by averaging the fluorescence over time intervals of 2 s and recording the integrated images. The fluorescence intensity reached its maximum in about 20 - 30 min after injection, presumably after monomerization inside erythrocyte membranes. The results are interpreted in terms of physical-chemical characteristics (e.g. hydrophilicity) and correlated with the photodynamically induced hemostasis in CAM blood vessels.

Improvement of fluorescence intensity of nitrogen vacancy centers in self-formed diamond microstructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furuyama, S.; Yaita, J.; Kondo, M.

2015-10-19

We present umbrella-shaped diamond microstructures with metal mirrors at the bottom in order to improve the amount of collected photons from nitrogen vacancy centers. The metal mirrors at the bottom are self-aligned to the umbrella-shaped diamond microstructures which are selectively grown through holes created on a metal mask. By the finite-difference time-domain simulations, we found that the umbrella-shaped microstructures, which have an effect similar to solid immersion lens, could collect photons more efficiently than bulk or pillar-shaped microstructures. Improvement of the fluorescence intensity by factors of from 3 to 5 is shown experimentally.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis

PubMed Central

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-01-01

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots. PMID:28714873

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis.

PubMed

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-07-15

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.

Fluorescence lifetime imaging of skin cancer

NASA Astrophysics Data System (ADS)

Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

2011-03-01

Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Pulvermacher, P. M.; Schultz, E.; Schell, K.

2000-01-01

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

Multiple functionalization of fluorescent nanoparticles for specific biolabeling and drug delivery of dopamine

NASA Astrophysics Data System (ADS)

Malvindi, Maria Ada; di Corato, Riccardo; Curcio, Annalisa; Melisi, Daniela; Rimoli, Maria Grazia; Tortiglione, Claudia; Tino, Angela; George, Chandramohan; Brunetti, Virgilio; Cingolani, Roberto; Pellegrino, Teresa; Ragusa, Andrea

2011-12-01

The development of fluorescent biolabels for specific targeting and controlled drug release is of paramount importance in biological applications due to their potential in the generation of novel tools for simultaneous diagnosis and treatment of diseases. Dopamine is a neurotransmitter involved in several neurological diseases, such as Parkinson's disease and attention deficit hyperactivity disorder (ADHD), and the controlled delivery of its agonists already proved to have beneficial effects both in vitro and in vivo. Here, we report the synthesis and multiple functionalization of highly fluorescent CdSe/CdS quantum rods for specific biolabeling and controlled drug release. After being transferred into aqueous media, the nanocrystals were made highly biocompatible through PEG conjugation and covered by a carbohydrate shell, which allowed specific GLUT-1 recognition. Controlled attachment of dopamine through an ester bond also allowed hydrolysis by esterases, yielding a smart nanotool for specific biolabeling and controlled drug release.The development of fluorescent biolabels for specific targeting and controlled drug release is of paramount importance in biological applications due to their potential in the generation of novel tools for simultaneous diagnosis and treatment of diseases. Dopamine is a neurotransmitter involved in several neurological diseases, such as Parkinson's disease and attention deficit hyperactivity disorder (ADHD), and the controlled delivery of its agonists already proved to have beneficial effects both in vitro and in vivo. Here, we report the synthesis and multiple functionalization of highly fluorescent CdSe/CdS quantum rods for specific biolabeling and controlled drug release. After being transferred into aqueous media, the nanocrystals were made highly biocompatible through PEG conjugation and covered by a carbohydrate shell, which allowed specific GLUT-1 recognition. Controlled attachment of dopamine through an ester bond also allowed

Clinical application of indocyanine green (ICG) fluorescent imaging of hepatoblastoma.

PubMed

Yamamichi, Taku; Oue, Takaharu; Yonekura, Takeo; Owari, Mitsugu; Nakahata, Kengo; Umeda, Satoshi; Nara, Keigo; Ueno, Takehisa; Uehara, Shuichiro; Usui, Noriaki

2015-05-01

Although the usefulness of intraoperative indocyanine green (ICG) fluorescent imaging for the resection of hepatocellular carcinoma has been reported, its usefulness for the resection of hepatoblastoma remains unclear. This study clarifies the feasibility of intraoperative ICG fluorescent imaging for the resection of hepatoblastoma. In three hepatoblastoma patients, a primary tumor, recurrent tumor, and lung metastatic lesions were intraoperatively examined using a near-infrared fluorescence imaging system after the preoperative administration of ICG. ICG fluorescent imaging was useful for the surgical navigation in hepatoblastoma patients. In the first case, the primary hepatoblastoma exhibited intense fluorescence during right hepatectomy, but no fluorescence was detected in the residual liver. In the second case, a recurrent tumor exhibited fluorescence between the residual liver and diaphragm. A complete resection of the residual liver, with a partial resection of the diaphragm, followed by liver transplantation was performed. In the third case with multiple lung metastases, each metastatic lesion showed positive fluorescence, and all were completely resected. These fluorescence-positive lesions were pathologically proven to be viable hepatoblastoma cells. Intraoperative ICG fluorescence imaging for patients with hepatoblastoma was feasible and useful for identifying small viable lesions and confirming that no remnant tumor remained after resection. Copyright © 2015 Elsevier Inc. All rights reserved.

Multiple Velocity Profile Measurements in Hypersonic Flows using Sequentially-Imaged Fluorescence Tagging

NASA Technical Reports Server (NTRS)

Bathel, Brett F.; Danehy, Paul M.; Inmian, Jennifer A.; Jones, Stephen B.; Ivey, Christopher B.; Goyne, Christopher P.

2010-01-01

Nitric-oxide planar laser-induced fluorescence (NO PLIF) was used to perform velocity measurements in hypersonic flows by generating multiple tagged lines which fluoresce as they convect downstream. For each laser pulse, a single interline, progressive scan intensified CCD camera was used to obtain separate images of the initial undelayed and delayed NO molecules that had been tagged by the laser. The CCD configuration allowed for sub-microsecond acquisition of both images, resulting in sub-microsecond temporal resolution as well as sub-mm spatial resolution (0.5-mm x 0.7-mm). Determination of axial velocity was made by application of a cross-correlation analysis of the horizontal shift of individual tagged lines. Quantification of systematic errors, the contribution of gating/exposure duration errors, and influence of collision rate on fluorescence to temporal uncertainty were made. Quantification of the spatial uncertainty depended upon the analysis technique and signal-to-noise of the acquired profiles. This investigation focused on two hypersonic flow experiments: (1) a reaction control system (RCS) jet on an Orion Crew Exploration Vehicle (CEV) wind tunnel model and (2) a 10-degree half-angle wedge containing a 2-mm tall, 4-mm wide cylindrical boundary layer trip. The experiments were performed at the NASA Langley Research Center's 31-inch Mach 10 wind tunnel.

Development of ultrasound-assisted fluorescence imaging of indocyanine green.

PubMed

Morikawa, Hiroyasu; Toyota, Shin; Wada, Kenji; Uchida-Kobayashi, Sawako; Kawada, Norifumi; Horinaka, Hiromichi

2017-01-01

Indocyanine green (ICG) accumulation in hepatocellular carcinoma means tumors can be located by fluorescence. However, because of light scattering, it is difficult to detect ICG fluorescence from outside the body. We propose a new fluorescence imaging method that detects changes in the intensity of ICG fluorescence by ultrasound-induced temperature changes. ICG fluorescence intensity decreases as the temperature rises. Therefore, it should theoretically be possible to detect tissue distribution of ICG using ultrasound to heat tissue, moving the point of ultrasound transmission, and monitoring changes in fluorescence intensity. A new probe was adapted for clinical application. It consisted of excitation light from a laser, fluorescence sensing through a light pipe, and heating by ultrasound. We applied the probe to bovine liver to image the accumulation of ICG. ICG emits fluorescence (820 nm) upon light irradiation (783 nm). With a rise in temperature, the fluorescence intensity of ICG decreased by 0.85 %/°C. The distribution of fluorescent ICG was detected using an ultrasonic warming method in a new integrated probe. Modulating fluorescence by changing the temperature using ultrasound can determine where ICG accumulates at a depth, highlighting its potential as a means to locate hepatocellular carcinoma.

Palus Somni - Anomalies in the correlation of Al/Si X-ray fluorescence intensity ratios and broad-spectrum visible albedos. [lunar surface mineralogy

NASA Technical Reports Server (NTRS)

Clark, P. E.; Andre, C. G.; Adler, I.; Weidner, J.; Podwysocki, M.

1976-01-01

The positive correlation between Al/Si X-ray fluorescence intensity ratios determined during the Apollo 15 lunar mission and a broad-spectrum visible albedo of the moon is quantitatively established. Linear regression analysis performed on 246 1 degree geographic cells of X-ray fluorescence intensity and visible albedo data points produced a statistically significant correlation coefficient of .78. Three distinct distributions of data were identified as (1) within one standard deviation of the regression line, (2) greater than one standard deviation below the line, and (3) greater than one standard deviation above the line. The latter two distributions of data were found to occupy distinct geographic areas in the Palus Somni region.

Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin.

PubMed

Nakashima, Kenichi; Yuda, Kazuki; Ozaki, Yukihiro; Noda, Isao

2003-11-01

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.

Fluorescent Applications to Crystallization

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

2006-01-01

By covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and tests with model proteins have shown that labeling u to 5 percent of the protein molecules does not affect the X-ray data quality obtained . The presence of the trace fluorescent label gives a number of advantages. Since the label is covalently attached to the protein molecules, it "tracks" the protein s response to the crystallization conditions. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a darker background. Non-protein structures, such as salt crystals, do not show up under fluorescent illumination. Crystals have the highest protein concentration and are readily observed against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. Preliminary tests, using model proteins, indicates that we can use high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that more rapid amorphous precipitation kinetics may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Experiments are now being carried out to test this approach using a wider range, of proteins. The trace fluorescently labeled crystals will also

Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation

PubMed Central

Macpherson, Lindsey J.; Zaharieva, Emanuela E.; Kearney, Patrick J.; Alpert, Michael H.; Lin, Tzu-Yang; Turan, Zeynep; Lee, Chi-Hon; Gallio, Marco

2015-01-01

Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system. PMID:26635273

Diurnal Variability in Chlorophyll-a, Carotenoids, CDOM and SO42− Intensity of Offshore Seawater Detected by an Underwater Fluorescence-Raman Spectral System

PubMed Central

Chen, Jing; Ye, Wangquan; Guo, Jinjia; Luo, Zhao; Li, Ying

2016-01-01

A newly developed integrated fluorescence-Raman spectral system (λex = 532 nm) for detecting Chlorophyll-a (chl-a), Chromophoric Dissolved Organic Matter (CDOM), carotenoids and SO42− in situ was used to successfully investigate the diurnal variability of all above. Simultaneously using the integration of fluorescence spectroscopy and Raman spectroscopy techniques provided comprehensive marine information due to the complementarity between the different excitation mechanisms and different selection rules. The investigation took place in offshore seawater of the Yellow Sea (36°05′40′′ N, 120°31′32′′ E) in October 2014. To detect chl-a, CDOM, carotenoids and SO42−, the fluorescence-Raman spectral system was deployed. It was found that troughs of chl-a and CDOM fluorescence signal intensity were observed during high tides, while the signal intensity showed high values with larger fluctuations during ebb-tide. Chl-a and carotenoids were influenced by solar radiation within a day cycle by different detection techniques, as well as displaying similar and synchronous tendency. CDOM fluorescence cause interference to the measurement of SO42−. To avoid such interference, the backup Raman spectroscopy system with λex = 785 nm was employed to detect SO42− concentration on the following day. The results demonstrated that the fluorescence-Raman spectral system has great potential in detection of chl-a, carotenoids, CDOM and SO42− in the ocean. PMID:27420071

Multiple nosocomial infections: a risk of modern intensive care.

PubMed

Leviten, D L; Shulman, S T

1980-03-01

Many components of modern medical care greatly predispose subjects to nosocomial infection. These include cancer chemotherapy, organ transplantation, immunosuppression, and intensive supportive care, particularly in conjunction with mechanical ventilatory support, invasive monitoring devices and prolonged central or peripheral intravenous therapy. The hazard of nosocomial infection associated with residence in a modern intensive care unit is dramatized by the case history of a near-drowning victim whose hospital course was complicated by an unusually large number and variety of nosocomial bacterial infections. Sixteen different bacterial organisms were isolated from cultures of blood, purulent thoracostomy tube drainage, or purulent tracheal secretions during the patient's prolonged hospital course. Factors which predisposed this patient to nosocomial infections included prolonged positive pressure mechanical ventilation, long-term broad spectrum antibiotics, indwelling arterial and central venous lines, violation of anatomic barriers by foreign bodies such as multiple thoracostomy tubes, and residence in an intensive care unit. This patient's case demonstrates that effective means to prevent nosocomial colonization and infection are urgently needed.

Method of determining the optimal dilution ratio for fluorescence fingerprint of food constituents.

PubMed

Trivittayasil, Vipavee; Tsuta, Mizuki; Kokawa, Mito; Yoshimura, Masatoshi; Sugiyama, Junichi; Fujita, Kaori; Shibata, Mario

2015-01-01

Quantitative determination by fluorescence spectroscopy is possible because of the linear relationship between the intensity of emitted fluorescence and the fluorophore concentration. However, concentration quenching may cause the relationship to become nonlinear, and thus, the optimal dilution ratio has to be determined. In the case of fluorescence fingerprint (FF) measurement, fluorescence is measured under multiple wavelength conditions and a method of determining the optimal dilution ratio for multivariate data such as FFs has not been reported. In this study, the FFs of mixed solutions of tryptophan and epicatechin of different concentrations and composition ratios were measured. Principal component analysis was applied, and the resulting loading plots were found to contain useful information about each constituent. The optimal concentration ranges could be determined by identifying the linear region of the PC score plotted against total concentration.

Differentiation of ocular fundus fluorophores by fluorescence lifetime imaging using multiple excitation and emission wavelengths

NASA Astrophysics Data System (ADS)

Hammer, M.; Schweitzer, D.; Schenke, S.; Becker, W.; Bergmann, A.

2006-10-01

Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH II provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.

Time-resolved fluorescence of thioredoxin single-tryptophan mutants: modeling experimental results with minimum perturbation mapping

NASA Astrophysics Data System (ADS)

Silva, Norberto D., Jr.; Haydock, Christopher; Prendergast, Franklyn G.

1994-08-01

The time-resolved fluorescence decay of single tryptophan (Trp) proteins is typically described using either a distribution of lifetimes or a sum of two or more exponential terms. A possible interpretation for this fluorescence decay heterogeneity is the existence of different isomeric conformations of Trp about its (chi) +1) and (chi) +2) dihedral angles. Are multiple Trp conformations compatible with the remainder of the protein in its crystallographic configuration or do they require repacking of neighbor side chains? It is conceivable that isomers of the neighbor side chains interconvert slowly on the fluorescence timescale and contribute additional lifetime components to the fluorescence intensity. We have explored this possibility by performing minimum perturbation mapping simulations of Trp 28 and Trp 31 in thioredoxin (TRX) using CHARMm 22. Mappings of Trp 29 and Trp 31 give the TRX Trp residue energy landscape as a function of (chi) +1) and (chi) +2) dihedral angles. Time-resolved fluorescence intensity and anisotropy decay of mutant TRX (W28F and W31F) are measured and interpreted in light of the above simulations. Relevant observables, like order parameters and isomerization rates, can be derived from the minimum perturbation maps and compared with experiment.

Differentiation of cancerous and normal brain tissue using label free fluorescence and Stokes shift spectroscopy

NASA Astrophysics Data System (ADS)

Zhou, Yan; Wang, Leana; Liu, Cheng-hui; He, Yong; Yu, Xinguang; Cheng, Gangge; Wang, Peng; Shu, Cheng; Alfano, Robert R.

2016-03-01

In this report, optical biopsy was applied to diagnose human brain cancer in vitro for the identification of brain cancer from normal tissues by native fluorescence and Stokes shift spectra (SSS). 77 brain specimens including three types of human brain tissues (normal, glioma and brain metastasis of lung cancers) were studied. In order to observe spectral changes of fluorophores via fluorescence, the selected excitation wavelength of UV at 300 and 340 nm for emission spectra and a different Stokes Shift spectra with intervals Δλ = 40 nm were measured. The fluorescence spectra and SSS from multiple key native molecular markers, such as tryptophan, collagen, NADH, alanine, ceroid and lipofuscin were observed in normal and diseased brain tissues. Two diagnostic criteria were established based on the ratios of the peak intensities and peak position in both fluorescence and SSS spectra. It was observed that the ratio of the spectral peak intensity of tryptophan (340 nm) to NADH (440 nm) increased in glioma, meningioma (benign), malignant meninges tumor, and brain metastasis of lung cancer tissues in comparison with normal tissues. The ratio of the SS spectral peak (Δλ = 40 nm) intensities from 292 nm to 366 nm had risen similarly in all grades of tumors.

Fluorescence excitation and imaging of single molecules near dielectric-coated and bare surfaces: a theoretical study.

PubMed

Axelrod, Daniel

2012-08-01

Microscopic fluorescent samples of interest to cell and molecular biology are commonly embedded in an aqueous medium near a solid surface that is coated with a thin film such as a lipid multilayer, collagen, acrylamide, or a cell wall. Both excitation and emission of fluorescent single molecules near film-coated surfaces are strongly affected by the proximity of the coated surface, the film thickness, its refractive index and the fluorophore's orientation. For total internal reflection excitation, multiple reflections in the film can lead to resonance peaks in the evanescent intensity versus incidence angle curve. For emission, multiple reflections arising from the fluorophore's near field emission can create a distinct intensity pattern in both the back focal plane and the image plane of a high aperture objective. This theoretical analysis discusses how these features can be used to report film thickness and refractive index, and fluorophore axial position and orientation. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Fluorescence lifetime imaging ophthalmoscopy.

PubMed

Dysli, Chantal; Wolf, Sebastian; Berezin, Mikhail Y; Sauer, Lydia; Hammer, Martin; Zinkernagel, Martin S

2017-09-01

Imaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases. Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Fluorescent-Magnetic-Biotargeting Multifunctional Nanobioprobes for Detecting and Isolating Multiple Types of Tumor Cells

PubMed Central

Song, Er-Qun; Hu, Jun; Wen, Cong-Ying; Tian, Zhi-Quan; Yu, Xu; Zhang, Zhi-Ling; Shi, Yun-Bo; Pang, Dai-Wen

2011-01-01

Fluorescent-magnetic-biotargeting multifunctional nanobioprobes (FMBMNs) have attracted great attention in recent years due to their increasing, important applications in biomedical research, clinical diagnosis, and biomedicine. We have previously developed such nanobioprobes for the detection and isolation of a single kind of tumor cells. Detection and isolation of multiple tumor markers or tumor cells from complex samples sensitively and with high efficiency is critical for the early diagnosis of tumors, especially malignant tumors or cancers, which will improve clinical diagnosis outcomes and help to select effective treatment approaches. Here, we expanded the application of the monoclonal antibody (mAb)-coupled FMBMNs for multiplexed assays. Multiple types of cancer cells, such as leukemia cells and prostate cancer cells, were detected and collected from mixed samples within 25 minutes by using a magnet and an ordinary fluorescence microscope. The capture efficiencies of mAb-coupled FMBMNs for the above mentioned two types of cells were 96% and 97% respectively. Furthermore, by using the mAb-coupled FMBMNs, specific and sensitive detection and rapid separation of a small number of spiked leukemia cells and prostate cancer cells in a large population of cultured normal cells (about 0.01% were tumor cells) were achieved simply and inexpensively without any sample pretreatment before cell analysis. Therefore, mAb-coupled multicolour FMBMNs may be used for very sensitive detection and rapid isolation of multiple cancer cells in biomedical research and medical diagnostics. PMID:21250650

Fluorescence Imaging Reveals Surface Contamination

NASA Technical Reports Server (NTRS)

Schirato, Richard; Polichar, Raulf

1992-01-01

In technique to detect surface contamination, object inspected illuminated by ultraviolet light to make contaminants fluoresce; low-light-level video camera views fluorescence. Image-processing techniques quantify distribution of contaminants. If fluorescence of material expected to contaminate surface is not intense, tagged with low concentration of dye.

Anomalous fluorescence enhancement and fluorescence quenching of graphene quantum dots by single walled carbon nanotubes.

PubMed

Das, Ruma; Rajender, Gone; Giri, P K

2018-02-07

We explore the mechanism of the fluorescence enhancement and fluorescence quenching effect of single walled carbon nanotubes (SWCNTs) on highly fluorescent graphene quantum dots (GQDs) over a wide range of concentrations of SWCNTs. At very low concentrations of SWCNTs, the fluorescence intensity of the GQDs is enhanced, while at higher concentrations, systematic quenching of fluorescence is observed. The nature of the Stern-Volmer plot for the latter case was found to be non-linear indicating a combined effect of dynamic and static quenching. The contribution of the dynamic quenching component was assessed through the fluorescence lifetime measurements. The contribution of static quenching is confirmed from the red shift of the fluorescence spectra of the GQDs after addition of SWCNTs. The fluorescence intensity is first enhanced at very low concentration due to improved dispersion and higher absorption by GQDs, while at higher concentration, the fluorescence of GQDs is quenched due to the complex formation and associated reduction of the radiative sites of the GQDs, which is confirmed from time-resolved fluorescence measurements. Laser confocal microscopy imaging provides direct evidence of the enhancement and quenching of fluorescence at low and high concentrations of SWCNTs, respectively. This study provides an important insight into tuning the fluorescence of GQDs and understanding the interaction between GQDs and different CNTs, which is important for bio-imaging and drug delivery applications.

Enhanced fluorescence microscope and its application

NASA Astrophysics Data System (ADS)

Wang, Susheng; Li, Qin; Yu, Xin

1997-12-01

A high gain fluorescence microscope is developed to meet the needs in medical and biological research. By the help of an image intensifier with luminance gain of 4 by 104 the sensitivity of the system can achieve 10-6 1x level and be 104 times higher than ordinary fluorescence microscope. Ultra-weak fluorescence image can be detected by it. The concentration of fluorescent label and emitting light intensity of the system are decreased as much as possible, therefore, the natural environment of the detected call can be kept. The CCD image acquisition set-up controlled by computer obtains the quantitative data of each point according to the gray scale. The relation between luminous intensity and output of CCD is obtained by using a wide range weak photometry. So the system not only shows the image of ultra-weak fluorescence distribution but also gives the intensity of fluorescence of each point. Using this system, we obtained the images of distribution of hypocrellin A (HA) in Hela cell, the images of Hela cell being protected by antioxidant reagent Vit. E, SF and BHT. The images show that the digitized ultra-sensitive fluorescence microscope is a useful tool for medical and biological research.

Chemical Environment Effects on K[beta]/K[alpha] Intensity Ratio: An X-Ray Fluorescence Experiment on Periodic Trends

ERIC Educational Resources Information Center

Durham, Chaney R.; Chase, Jeffery M.; Nivens, Delana A.; Baird, William H.; Padgett, Clifford W.

2011-01-01

X-ray fluorescence (XRF) data from an energy-dispersive XRF instrument were used to investigate the chlorine K[alpha] and K[beta] peaks in several group 1 salts. The ratio of the peak intensity is sensitive to the local chemical environment of the chlorine atoms studied in this experiment and it shows a periodic trend for these salts. (Contains 1…

Impact of high-intensity concurrent training on cardiovascular risk factors in persons with multiple sclerosis - pilot study.

PubMed

Keytsman, Charly; Hansen, Dominique; Wens, Inez; O Eijnde, Bert

2017-10-27

High-intensity concurrent training positively affects cardiovascular risk factors. Because this was never investigated in multiple sclerosis, the present pilot study explored the impact of this training on cardiovascular risk factors in this population. Before and after 12 weeks of high-intense concurrent training (interval and strength training, 5 sessions per 2 weeks, n = 16) body composition, resting blood pressure and heart rate, 2-h oral glucose tolerance (insulin sensitivity, glycosylated hemoglobin, blood glucose and insulin concentrations), blood lipids (high- and low-density lipoprotein, total cholesterol, triglyceride levels) and C-reactive protein were analyzed. Twelve weeks of high-intense concurrent training significantly improved resting heart rate (-6%), 2-h blood glucose concentrations (-13%) and insulin sensitivity (-24%). Blood pressure, body composition, blood lipids and C-reactive protein did not seem to be affected. Under the conditions of this pilot study, 12 weeks of concurrent high-intense interval and strength training improved resting heart rate, 2-h glucose and insulin sensitivity in multiple sclerosis but did not affect blood C-reactive protein levels, blood pressure, body composition and blood lipid profiles. Further, larger and controlled research investigating the effects of high-intense concurrent training on cardiovascular risk factors in multiple sclerosis is warranted. Implications for rehabilitation High-intensity concurrent training improves cardiovascular fitness. This pilot study explores the impact of this training on cardiovascular risk factors in multiple sclerosis. Despite the lack of a control group, high-intense concurrent training does not seem to improve cardiovascular risk factors in multiple sclerosis.

Wireless implantable electronic platform for chronic fluorescent-based biosensors.

PubMed

Valdastri, Pietro; Susilo, Ekawahyu; Förster, Thilo; Strohhöfer, Christof; Menciassi, Arianna; Dario, Paolo

2011-06-01

The development of a long-term wireless implantable biosensor based on fluorescence intensity measurement poses a number of technical challenges, ranging from biocompatibility to sensor stability over time. One of these challenges is the design of a power efficient and miniaturized electronics, enabling the biosensor to move from bench testing to long term validation, up to its final application in human beings. In this spirit, we present a wireless programmable electronic platform for implantable chronic monitoring of fluorescent-based autonomous biosensors. This system is able to achieve extremely low power operation with bidirectional telemetry, based on the IEEE802.15.4-2003 protocol, thus enabling over three-year battery lifetime and wireless networking of multiple sensors. During the performance of single fluorescent-based sensor measurements, the circuit drives a laser diode, for sensor excitation, and acquires the amplified signals from four different photodetectors. In vitro functionality was preliminarily tested for both glucose and calcium monitoring, simply by changing the analyte-binding protein of the biosensor. Electronics performance was assessed in terms of timing, power consumption, tissue exposure to electromagnetic fields, and in vivo wireless connectivity. The final goal of the presented platform is to be integrated in a complete system for blood glucose level monitoring that may be implanted for at least one year under the skin of diabetic patients. Results reported in this paper may be applied to a wide variety of biosensors based on fluorescence intensity measurement.

Analysis of rainwater dissolved organic carbon compounds using fluorescence spectrophotometry

NASA Astrophysics Data System (ADS)

Muller, Catherine L.; Baker, Andy; Hutchinson, Robert; Fairchild, Ian J.; Kidd, Chris

Global rainwater dissolved organic carbon (DOC) flux was recently estimated as 430 × 10 12 g C yr -1, yet little is known about the wide range of chemical compounds present, their sources, temporal patterns of variation, and the subsequent impact on climate and the environment. Precipitation events were sampled in Birmingham, UK between April 2005 and May 2007. Rainwater DOC compounds were analysed using fluorescence spectrophotometry. Three fluorophores were identified: HUmic-LIke Substances (HULIS), TYrosine-LIke Substances (TYLIS) and TRYptophan-LIke Substances (TRYLIS). Peak fluorescence intensities and locations for each substance were examined, and their variations with various meteorological parameters were investigated. The mean HULIS fluorescence intensity from all events was 209 a.u. (with sample fluorescence ranging from 37 a.u. to 995 a.u); mean fluorescence intensity was 469 a.u. (214-988 a.u) and 265 a.u. (50-876 a.u.) for TYLIS and TRYLIS, respectively. Results indicate that highest HULIS fluorescence intensities are experienced during convective events and events of continental origin, suggesting terrestrial/anthropogenic sources. Under well-mixed conditions, HULIS fluorescence intensity decreases, whereas during low wind speed, stagnation of the atmosphere results in higher fluorescence intensities, attributed to a build up of localised sources, particularly anthropogenic. TYLIS and TRYLIS did not show any significant trends for the meteorological variables. Fluorescence spectrophotometry is a fast, non-invasive technique which is demonstrated to be a powerful means of fingerprinting rainfall DOC compounds in real time for small sample volumes.

Surface Coverage and Structure of Mixed DNA/Alkylthiol Monolayers on Gold: Characterization by XPS, NEXAFS, and Fluorescence Intensity Measurements

PubMed Central

Lee, Chi-Ying; Gong, Ping; Harbers, Gregory M.; Grainger, David W.; Castner, David G.; Gamble, Lara J.

2006-01-01

Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s → π* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex

Efficient HOMO-LUMO separation by multiple resonance effect toward ultrapure blue thermally activated delayed fluorescence

NASA Astrophysics Data System (ADS)

Hatakeyama, Takuji; Ikuta, Toshiaki; Shiren, Kazushi; Nakajima, Kiichi; Nomura, Shintaro; Ni, Jingping

2016-09-01

Organic light-emitting diodes (OLEDs) play an important role in the new generation of flat-panel displays. Conventional OLEDs employing fluorescent materials together with triplet-triplet annihilation suffer from a relatively low internal quantum efficiency (IQE) of 62.5%. On the other hand, the IQE of OLEDs employing phosphorescent or thermally activated delayed fluorescence (TADF) materials can reach 100%. However, these materials exhibit very broad peaks with a full-width at half-maximum (FWHM) of 70-100 nm and cannot satisfy the color-purity requirements for displays. Therefore, the latest commercial OLED displays employ blue fluorescent materials with a relatively low IQE, and efficient blue emitters with a small FWHM are highly needed. In our manuscript, we present organic molecules that exhibit ultrapure blue fluorescence based on TADF. These molecules consist of three benzene rings connected by one boron and two nitrogen atoms, which establish a rigid polycyclic framework and significant localization of the highest occupied and lowest unoccupied molecular orbitals by a multiple resonance effect. An OLED device based on the new emitter exhibits ultrapure blue emission at 467 nm with an FWHM of 28 nm, Commission Internationale de l'Eclairage (CIE) coordinates of (0.12, 0.13), and an IQE of 100%, which represent record-setting performance for blue OLED devices.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

2006-01-01

We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider

A Passive Method for Detecting Vegetation Stress from Orbit: Chlorophyll Fluorescence Spectra from Fraunhofer Lines

NASA Technical Reports Server (NTRS)

Theisen, Arnold F.

2000-01-01

Solar-stimulated chlorophyll fluorescence measured with the Fraunhofer line depth method has correlated well with vegetation stress in previous studies. However, the instruments used in those studies were limited to a single solar absorption line (e.g. 656.3 nm), obviating the red/far-red ratio (R/FR) method. Optics and detector technology have reached the level whereby multiple, very narrow Fraunhofer lines are resolvable. Thirteen such lines span the visible spectrum in the red to far-red region where chlorophyll fluorescence occurs. Fluorescence intensities at the 13 Fraunhofer line wavelengths were used to model emission spectra. The source data were collected for summer and fall bean crops (Phaseolus vulgaris L.) subjected to various levels of nitrogen fertilization. The intensities were adjusted to account for Fraunhofer line depth and atmospheric transmittance. Multiple R/FR fluorescence ratios, calculated from the modeled fluorescence spectra, correlated strongly with leaf chlorophyll concentration and well with applied nitrogen. The ratio yielding the best correlation with chlorophyll utilized red fluorescence at the 694.5 nm Fraunhofer line and farred fluorescence at the 755.6 nm Fraunhofer line. Twenty R/FR ratios, each evaluated for the maximum differential between low and high (optimal) nitrogen treatments, ranked higher in some cases and lower in others, possibly related to the time of year the crops were grown and the stage of growth of the crops. Ratios with 728.9 nm and 738.9 nm in the denominator consistently ranked in the lowest and next lowest quartile, respectively. Ratios of the 656.3 nm Fraunhofer line and the 755.6 nm line consistently ranked highest for the summer crop. Ratios with 755.6 nm in the denominator ranked in the upper quartile for 10 out of 12 measurement dates. Differences in ratio ranking indicate that physiological conditions may be estimated using selected ratios of Fraunhofer lines within the context of R/FR analysis. A

Hydrogen-Bond and Supramolecular-Contact Mediated Fluorescence Enhancement of Electrochromic Azomethines.

PubMed

Wałęsa-Chorab, Monika; Tremblay, Marie-Hélène; Skene, William G

2016-08-01

An electronic push-pull fluorophore consisting of an intrinsically fluorescent central fluorene capped with two diaminophenyl groups was prepared. An aminothiophene was conjugated to the two flanking diphenylamines through a fluorescent quenching azomethine bond. X-ray crystallographic analysis confirmed that the fluorophore formed multiple intermolecular supramolecular bonds. It formed two hydrogen bonds involving a terminal amine, resulting in an antiparallel supramolecular dimer. Hydrogen bonding was also confirmed by FTIR and NMR spectroscopic analyses, and further validated theoretically by DFT calculations. Intrinsic fluorescence quenching modes could be reduced by intermolecular supramolecular contacts. These contacts could be engaged at high concentrations and in thin films, resulting in fluorescence enhancement. The fluorescence of the fluorophore could also be restored to an intensity similar to its azomethine-free counterpart with the addition of water in >50 % v/v in tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), and acetonitrile. The fluorophore also exhibited reversible oxidation and its color could be switched between yellow and blue when oxidized. Reversible electrochemically mediated fluorescence turn-off on turn-on was also possible. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma

PubMed Central

Meier, Jeremy D.; Xie, Hongtao; Sun, Yang; Sun, Yinghua; Hatami, Nisa; Poirier, Brian; Marcu, Laura; Farwell, D. Gregory

2011-01-01

OBJECTIVE 1) Determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract. 2) Evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN Cross-sectional study. SETTING University-based medical center. SUBJECTS AND METHODS Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700 ps pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared to the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. RESULTS Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than the normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360~660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7±0.06 ns for normal and 1.3±0.06 ns for tumor, P=0.0025), and the Laguerre coefficients, LEC-2 (i.e., at 460 nm: 0.135±0.001 for normal and 0.155±0.007 for tumor, P<0.05). CONCLUSION These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a non-invasive diagnostic technique for HNSCC. PMID:20493355

Kα resonance fluorescence in Al, Ti, Cu and potential applications for X-ray sources

NASA Astrophysics Data System (ADS)

Nahar, Sultana N.; Pradhan, Anil K.

2015-04-01

The Kα resonance fluorescence (RFL) effect via photoabsorptions of inner shell electrons as the element goes through multiple ionization states is studied. We demonstrate that the resonances observed recently in Kα (1s-2p) fluorescence in aluminum plasmas by using a high-intensity X-ray free-electron laser [1] are basically K-shell resonances in hollow atoms going through multiple ionization states at resonant energies as predicted earlier for gold and iron ions [2]. These resonances are formed below the K-shell ionization edge and shift toward higher energies with ionization states, as observed. Fluorescence emission intensities depend on transition probabilities for each ionization stage of the given element for all possible Kα (1 s → 2 p) transition arrays. The present calculations for resonant photoabsorptions of Kα photons in Al have reproduced experimentally observed features. Resonant cross sections and absorption coefficients are presented for possible observation of Kα RFL in the resonant energy ranges of 4.5-5.0 keV for Ti ions and 8.0-8.7 keV for Cu ions respectively. We suggest that theoretically the Kα RFL process may be driven to enhance the Auger cycle by a twin-beam monochromatic X-ray source, tuned to the K-edge and Kα energies, with potential applications such as the development of narrow-band biomedical X-ray devices.

Fluorescence from polystyrene - Photochemical processes in polymeric systems, 7

NASA Technical Reports Server (NTRS)

Gupta, M. C.; Gupta, A.

1983-01-01

Results are presented for measurements of the fluorescence spectra of polystyrene in dilute solution and in pure solid films. It is determined that a major potential source of experimental error is the concurrent photooxidative degradation in air which may obscure fluorescence emission from monomeric sites in solid films at 25 C. The fluorescence spectra of oriented films are evaluated in terms of the monomer to excimer fluorescence intensity ratio and the excimer 'red shift'. The monomer to excimer fluorescence intensity ratio is determined to be significantly higher in fluid solution than in solid film.

Methods on observation of fluorescence micro-imaging for microalgae

NASA Astrophysics Data System (ADS)

Ou, Lin; Zhuang, Hui-ru; Chen, Rong; Lei, Jin-pin; Liao, Xiao-hua; Lin, Wen-suo

2007-11-01

Objective: Auto-fluorescence micro-imaging of microalgae are observed by using of laser scanning confocal microscopy (LSCM) and fluorescence microscopy, so as to investigate the effect of auto fluorescence alteration on growth of irradiated microalgae irradiated, meanwhile, the method of microalgae cells stained also to be studied. Methods: Platymonas subcordiformis, Phaeodactylum tricormutum and Isochyrsis zhanjiangensis cells are stained with acridine orange, and observed by fluorescence microscopy; the three types microalgae mentioned above are irradiated by Nd:YAP laser with 10w at 1341nm, irradiating time:12s, 30s, 35s and 55s, than to be cultured 6 days, and the auto fluorescence images and fluorescence spectra of algae cells are obtained by LSCM on lambda scan mode, at excitation 488nm (Ar + laser). Results: It is showed that the shapes and the structural features of microalgae cells stained can be seen clearly, and the cytoplasm and nucleus also can be observed. The chloroplasts in cell is bigger on promoting effects, conversely, it is to be mutilated, deformation and shrink. Contrast to the CK, the peak positions of fluorescence of algae cells irradiated is similar to the whole while the peak light intensity alters. On irradiation of promoting dose, however, the auto fluorescence intensity is enhanced more than control. Conclusions: The method of cell stained can be used to observed genetic material in microalgae. There are obvious effects for laser irradiating to chloroplasts in cells, the bigger chloroplasts the greater fluorescence intensity. Physiological incentive effects of microalgae irradiated can be given expression on fluorescence characteristics and fluorescence intensity alteration of cells.

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

PubMed

Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki

2013-07-29

We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

Fluorescent intensity-based differential counting of FITC-doped silica nanoparticles: applications of CD4+ T-cell detection in microchip-type flowcytometers

NASA Astrophysics Data System (ADS)

Yun, Hoyoung; Bang, Hyunwoo; Lee, Won Gu; Lim, Hyunchang; Park, Junha; Lee, Joonmo; Riaz, Asif; Cho, Keunchang; Chung, Chanil; Han, Dong-Chul; Chang, Jun Keun

2007-12-01

Although CD4+ T-cells are an important target of HIV detection, there have been still major problems in making a diagnosis and monitoring in the third world and the region with few medical facilities. Then, it is necessary to use portable diagnosis devices at low cost when you put an enumeration of CD4+ T-cells. In general, the counting of CD4 below 200cells/uL makes it necessary to initiate antiretroviral treatment in adults (over 13 years old). However, lymphocyte subsets (including CD4 counts) of infants and young children are higher than those of adults. This fact shows the percentage of CD4+ T-cells of blood subsets, i.e., CD4/CD45%, CD4/CD8% or CD4/CD3% means a more reliable indicator of HIV infection than absolute counts in children. To know the percentage of CD4+ T-cell by using two fluorescent dyes of different emission wavelength, at least, one laser and two PMT detectors are in general needed. Then, it is so hard to develop a portable device like a 'toaster size' because this makes such a device more complex including many peripheral modules. In this study, we developed a novel technique to control the intensity of fluorescent dye-doped silica nanoparticles. I synthesized FITC-doped silica nanoparticles conjugated CD4 antibody 10 times brighter than FITC-conjugated CD45 antibody. With the difference of intensity of two fluorescent dyes, we measured two parameters by using only a single detector and laser. Most experiments were achieved with uFACS (microfabricated fluorescence-activated cell sorter) on an inverted microscope (IX71, Olympus). In conclusion, this method enables us to discriminate the difference between CD4 and CD45 in an intensity domain simultaneously. Furthermore, this technique would make it possible develop much cheaper and smaller devices which can count the number of CD4 T-cells.

Boundary segmentation for fluorescence microscopy using steerable filters

NASA Astrophysics Data System (ADS)

Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2017-02-01

Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

Spectral line discriminator for passive detection of fluorescence

NASA Technical Reports Server (NTRS)

Kebabian, Paul L. (Inventor)

1996-01-01

A method and apparatus for detecting fluorescence from sunlit plants is based on spectral line discrimination using the A-band and B-band absorption of atmospheric oxygen. Light from a plant including scattered sunlight and the fluorescence from chlorophyll is passed through a chopper into a cell containing low-pressure, high-purity oxygen. A-band or B-band wavelengths present in the light are absorbed by the oxygen in the cell. When the chopper is closed, the absorbed light is remitted as fluorescence into a detector. The intensity of the fluorescence from the oxygen is proportional to the intensity of fluorescence from the plant.

Ultrasound Induced Fluorescence of Nanoscale Liposome Contrast Agents

PubMed Central

Zhang, Qimei; Morgan, Stephen P.; O’Shea, Paul; Mather, Melissa L.

2016-01-01

A new imaging contrast agent is reported that provides an increased fluorescent signal upon application of ultrasound (US). Liposomes containing lipids labelled with pyrene were optically excited and the excimer fluorescence emission intensity was detected in the absence and presence of an ultrasound field using an acousto-fluorescence setup. The acousto-fluorescence dynamics of liposomes containing lipids with pyrene labelled on the fatty acid tail group (PyPC) and the head group (PyPE) were compared. An increase in excimer emission intensity following exposure to US was observed for both cases studied. The increased intensity and time constants were found to be different for the PyPC and PyPE systems, and dependent on the applied US pressure and exposure time. The greatest change in fluorescence intensity (130%) and smallest rise time constant (0.33 s) are achieved through the use of PyPC labelled liposomes. The mechanism underlying the observed increase of the excimer emission intensity in PyPC labelled liposomes is proposed to arise from the “wagging” of acyl chains which involves fast response and requires lower US pressure. This is accompanied by increased lipid lateral diffusivity at higher ultrasound pressures, a mechanism that is also active in the PyPE labelled liposomes. PMID:27467748

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis

PubMed Central

Cabrita, Marisa; Bekman, Evguenia; Braga, José; Rino, José; Santus, Renè; Filipe, Paulo L.; Sousa, Ana E.; Ferreira, João A.

2017-01-01

We propose a novel single-deoxynucleoside-based assay that is easy to perform and provides accurate values for the absolute length (in units of time) of each of the cell cycle stages (G1, S and G2/M). This flow-cytometric assay takes advantage of the excellent stoichiometric properties of azide-fluorochrome detection of DNA substituted with 5-ethynyl-2′-deoxyuridine (EdU). We show that by pulsing cells with EdU for incremental periods of time maximal EdU-coupled fluorescence is reached when pulsing times match the length of S phase. These pulsing times, allowing labelling for a full S phase of a fraction of cells in asynchronous populations, provide accurate values for the absolute length of S phase. We characterized additional, lower intensity signals that allowed quantification of the absolute durations of G1 and G2 phases. Importantly, using this novel assay data on the lengths of G1, S and G2/M phases are obtained in parallel. Therefore, these parameters can be estimated within a time frame that is shorter than a full cell cycle. This method, which we designate as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, was successfully applied to cell types with distinctive cell cycle features and shows excellent agreement with established methodologies for analysis of cell cycle kinetics. PMID:28465489

Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis.

PubMed

Schulze, Philipp; Ludwig, Martin; Belder, Detlev

2008-12-01

A high intensity 266 nm continuous wave (cw-) laser developed for material processing was utilised as an excitation source for sensitive native fluorescence detection of unlabelled compounds in MCE. This 120 mW laser was attached via an optical fibre into a commercial epifluorescence microscope. With this MCE set-up we evaluated the impact of laser power on the S/N of aromatic compounds as well as of proteins. Compared with a previous work which used a 4 mW pulsed laser for excitation, improved S/N for small aromatics and to a lesser extent for proteins could be attained. The LOD of the system was determined down to 24 ng/mL for serotonin (113 nM), 24 ng/mL for propranolol (81 nM), 80 ng/mL for tryptophan (392 nM) and 80 ng/mL for an aromatic diol (475 nM). Sensitive protein detection was obtained at concentrations of 5 microg/mL for lysocyme, trypsinogen and chymotrypsinogen (340, 208 and 195 nM, respectively). Finally, a comparison of the cw- with a pulsed 266 nm laser, operating at the same average power, showed a higher attainable sensitivity of the cw-laser. This can be attributed to fluorescence saturation and photobleaching effects of the pulsed laser at high pulse energies.

Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged Pseudomonas fluorescens A506

PubMed Central

Lowder, M.; Unge, A.; Maraha, N.; Jansson, J. K.; Swiggett, J.; Oliver, J. D.

2000-01-01

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5°C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30°C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30°C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50°C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost. PMID

Uptake of Fluorescent Gentamicin by Peripheral Vestibular Cells after Systemic Administration

PubMed Central

Liu, Jianping; Kachelmeier, Allan; Dai, Chunfu; Li, Hongzhe; Steyger, Peter S.

2015-01-01

Objective In addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours. Results Low intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs’ sensory cells. Control vestibular tissues exposed to Dulbecco’s phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence. Conclusions All peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may

Novel functionalized fluorescent polymeric nanoparticles for immobilization of biomolecules

NASA Astrophysics Data System (ADS)

Jain, Swati; Chattopadhyay, Sruti; Jackeray, Richa; Abid, C. K. V. Zainul; Singh, Harpal

2013-07-01

Novel, size controlled fluorescent polymeric nanoparticles (FPNP) were synthesized having acetoacetoxy functionality on the surface for immobilization of biomolecules which can be utilized as biomarkers and labels in fluoroimmunoassays. Core-shell nanoparticles of poly(styrene, St-methyl methacrylate, MMA-acetoacetoxy ethyl methacrylate, AAEM), stabilized by various concentrations of surfactant, sodium lauryl sulphate (SLS), were obtained by facile miniemulsion co-polymerization encapsulated with pyrene molecules in their hydrophobic core. Analytical, spectroscopic and imaging characterization techniques revealed the formation of stable, monodisperse, spherical nano sized particles exhibiting high luminescence properties. Particles with 1% SLS (S1) showed good dispersion stability and fluorescence intensity and were chosen as ideal candidates for further immobilization studies. Steady state fluorescence studies showed 10 times higher fluorescence intensity of S1 nanoparticles than that of pyrene solution in solvent-toluene at the same concentration. Environmental factors such as pH, ionic strength and time were found to have no effect on fluorescence intensity of FPNPs. Surface β-di-ketone groups were utilized for the covalent immobilization of enzyme conjugated antibodies without any activation or pre-treatment of nanoparticles.Novel, size controlled fluorescent polymeric nanoparticles (FPNP) were synthesized having acetoacetoxy functionality on the surface for immobilization of biomolecules which can be utilized as biomarkers and labels in fluoroimmunoassays. Core-shell nanoparticles of poly(styrene, St-methyl methacrylate, MMA-acetoacetoxy ethyl methacrylate, AAEM), stabilized by various concentrations of surfactant, sodium lauryl sulphate (SLS), were obtained by facile miniemulsion co-polymerization encapsulated with pyrene molecules in their hydrophobic core. Analytical, spectroscopic and imaging characterization techniques revealed the formation of stable

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma.

PubMed

Meier, Jeremy D; Xie, Hongtao; Sun, Yang; Sun, Yinghua; Hatami, Nisa; Poirier, Brian; Marcu, Laura; Farwell, D Gregory

2010-06-01

The objectives of this study were to 1) determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract, and 2) evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). Cross-sectional study. University-based medical center. Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700-picosecond pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared with the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than that of normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360 to approximately 660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7 +/- 0.06 ns [not significant] for normal and 1.3 +/- 0.06 ns for tumor, P = 0.0025) and the second-order Laguerre expansion coefficient (LEC-2) (i.e., at 460 nm: 0.135 +/- 0.001 for normal and 0.155 +/- 0.007 for tumor, P < 0.05). These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a noninvasive diagnostic technique for HNSCC. Copyright 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.

The Rate Constant for Fluorescence Quenching

ERIC Educational Resources Information Center

Legenza, Michael W.; Marzzacco, Charles J.

1977-01-01

Describes an experiment that utilizes fluorescence intensity measurements from a Spectronic 20 to determine the rate constant for the fluorescence quenching of various aromatic hydrocarbons by carbon tetrachloride in an ethanol solvent. (MLH)

Measurement of protein-like fluorescence in river and waste water using a handheld spectrophotometer.

PubMed

Baker, Andy; Ward, David; Lieten, Shakti H; Periera, Ryan; Simpson, Ellie C; Slater, Malcolm

2004-07-01

Protein-like fluorescence intensity in rivers increases with increasing anthropogenic DOM inputs from sewerage and farm wastes. Here, a portable luminescence spectrophotometer was used to investigate if this technology could be used to provide both field scientists with a rapid pollution monitoring tool and process control engineers with a portable waste water monitoring device, through the measurement of river and waste water tryptophan-like fluorescence from a range of rivers in NE England and from effluents from within two waste water treatment plants. The portable spectrophotometer determined that waste waters and sewerage effluents had the highest tryptophan-like fluorescence intensity, urban streams had an intermediate tryptophan-like fluorescence intensity, and the upstream river samples of good water quality the lowest tryptophan-like fluorescence intensity. Replicate samples demonstrated that fluorescence intensity is reproducible to +/- 20% for low fluorescence, 'clean' river water samples and +/- 5% for urban water and waste waters. Correlations between fluorescence measured by the portable spectrophotometer with a conventional bench machine were 0.91; (Spearman's rho, n = 143), demonstrating that the portable spectrophotometer does correlate with tryptophan-like fluorescence intensity measured using the bench spectrophotometer.

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

PubMed Central

Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2014-01-01

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding. PMID:24886825

Medium effects on fluorescence of ciprofloxacin hydrochloride

NASA Astrophysics Data System (ADS)

Yang, Rui; Fu, Yan; Li, Long-Di; Liu, Jia-Ming

2003-10-01

The medium (pH, organic solvents, cyclodextrin (CD) or surfactants) effects on the fluorescence of ciprofloxacin hydrochloride (CPFX·HCl) were studied in detail. It is found that the three acid constants of ciprofloxacin (CPFX) are near to each other. Therefore the relation curve between pH and fluorescence intensity has no strident change and keeps relative stable in the pH range of 2-7. When pH was in the range of 5.5-6.0, the fluorescence intensity of CPFX reached the max. The kind and amount of organic solvent added to the luminescent system have various effects. Ethanol quenched fluorescence and the fluorescence excitation wavelength is red shift at first and then blue shift. Acetone has complicated effects on the fluorescence properties of CPFX·HCl solution. The experiment result shows that acetone is really a quencher when its volume content in the system is from 0 to 20%, but when its content is 90%, the signal intensity is unexpectedly one and a half times as much as that of no acetone. This means that there is a strong interaction between the acetone and CPFX; CPFX·H + could be included into the γ-CD but the capping effect is not notable. The effect of cationic surfactant cetyltrimethylammonium bromide and non-ionic surfactant TX-100 and TX-80 on CPFX fluorescence was unimpressive, but the anionic surfactant's effect is aberrant. The fluorescence intensity of CPFX·HCl solution experiences three stages of increasing, decreasing and increasing in turn, as sodium dodecyl sulfate is adding gradually. But for sodium lauryl sulfonate, there are only two stages of decreasing and increasing with the concentration increasing. It is problematic to illustrate clearly the effect mechanism of acetone and anionic surfactant at present. Undoubtedly, the experimental results in this paper should be useful in practice works and the research is worth studying still further.

Simultaneous fluorescent detection of multiple metal ions based on the DNAzymes and graphene oxide.

PubMed

Yun, Wen; Wu, Hong; Liu, Xingyan; Fu, Min; Jiang, Jiaolai; Du, Yunfeng; Yang, Lizhu; Huang, Yu

2017-09-15

A novel fluorescent detection strategy for simultaneous detection of Cu 2+ , Pb 2+ and Mg 2+ based on DNAzyme branched junction structure with three kinds of DNAzymes and graphene oxide (GO) was presented. Three fluorophores labeled DNA sequences consisted with enzyme-strand (E-DNA) and substrate strand (S-DNA) were annealed to form DNAzyme branched junction structure. In the presence of target metal ion, the DNAzyme was activated to cleave the fluorophore labeled S-DNA. The S-DNA fragments were released and adsorbed onto GO surface to quench the fluorescent signal. The detection limit was calculated to be 1 nM for Cu 2+ , 200 nM for Mg 2+ , and 0.3 nM for Pb 2+ , respectively. This strategy was successfully used for simultaneous detection of Cu 2+ , Mg 2+ and Pb 2+ in human serum. Moreover, it had potential application for simultaneous detection of multiple metal ions in environmental and biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Iterative Correction Scheme Based on Discrete Cosine Transform and L1 Regularization for Fluorescence Molecular Tomography With Background Fluorescence.

PubMed

Zhang, Jiulou; Shi, Junwei; Guang, Huizhi; Zuo, Simin; Liu, Fei; Bai, Jing; Luo, Jianwen

2016-06-01

High-intensity background fluorescence is generally encountered in fluorescence molecular tomography (FMT), because of the accumulation of fluorescent probes in nontarget tissues or the existence of autofluorescence in biological tissues. The reconstruction results are affected or even distorted by the background fluorescence, especially when the distribution of fluorescent targets is relatively sparse. The purpose of this paper is to reduce the negative effect of background fluorescence on FMT reconstruction. After each iteration of the Tikhonov regularization algorithm, 3-D discrete cosine transform is adopted to filter the intermediate results. And then, a sparsity constraint step based on L1 regularization is applied to restrain the energy of the objective function. Phantom experiments with different fluorescence intensities of homogeneous and heterogeneous background are carried out to validate the performance of the proposed scheme. The results show that the reconstruction quality can be improved with the proposed iterative correction scheme. The influence of background fluorescence in FMT can be reduced effectively because of the filtering of the intermediate results, the detail preservation, and noise suppression of L1 regularization.

Interpretation of the fluorescence signatures from vegetation

NASA Astrophysics Data System (ADS)

Buschmann, C.

Vegetation emits fluorescence as part of the energy taken up by absorption %of solar radiation from UV to the visible. This fluorescence consists of light with low intensity (only few percents of the reflected light) emitted from the leaves. The fluorescence emission of a green leaf is characterized by four bands with maxima in the blue (440 nm), green (520 nm), red (690 nm) and far red (740 nm) spectral region. The intensity of fluorescence in the maxima of the emission spectrum varies depending on the following six basic parameters which must be taken into account for the interpretation of fluorescence signatures from vegetation: (a) content of the fluorophores (ferulic acid, chlorophyll a), (b) temperature of the leaf, (c) penetration of excitation light into the leaf, (d) emission of fluorescence from the leaf (re-absorption inside the leaf tissue), (e) photosynthetic activity of the leaf, (f) non-radiative decay (heat production) parallel to the fluorescence The ratios between the intensities of the maxima (F440/F690, F440/F520, F690/F740) are used as characteristic fluorescence parameter. The wide range of changes of these ratios caused by differences in the leaf tissue (aerial interspaces, variegated/homogeneous green leaves), various types of stress (UV, photoinhibition, sun exposure, heat, water deficiency, N-deficiency) and chemicals (inhibitors, fertilizers) can be explained by changes of the six basic parameters. It will be shown that the interpretation of the fluorescence signatures, in most cases, must be based on a complex consideration of more than one of the basic parameters.

Fluorescent and high intensity discharge lamp use in chambers and greenhouses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Langhans, R.W.

1994-12-31

Fluorescent and High Intensity Discharge lamps have opened up great opportunities for researchers to study plant growth under controlled environment conditions and for commercial growers to increase plant production during low/light periods. This report describes the advantages and disadvantages of using each lamp in growth chambers, growth rooms and greenhouses. Growth Chambers are small (3m x 4/m and smaller) walk-in or reach-in enclosures with programmable, accurate temperature, relative humidity (RH) and irradiance control over wide ranges. The intent of growth chambers was to replicate sunlight conditions and transfer research results directly to the greenhouse or outside. It was realized thatmore » sunlight and outside conditions could not be mimicked. Growth chambers are also used to study irradiance and spectral fluxes. Growth Rooms are usually large rooms (larger than 3m x 4m) with only lamp irradiance, but providing relatively limited ranges of environmental control (i.e., 10 to 30 C temperature, 50 to 90% RH and ambient to 1000 ppm CO{sub 2}), and commonly independent of outside conditions. Irradiance requirements for growth rooms are similar to those of growth chambers. Growth rooms are also used for growing a large number of plants in a uniform standard environment condition and in commercial horticulture for tissue culture, seed germination (plugs) and seedling growth. Greenhouses are designed to allow maximum sunlight penetration through the structure. Initially greenhouses were used to extend the growing season. Then as heating systems, and cooling systems improved, they were used year round. Low light during the winter months reduced plant growth, but with the advent of efficient lamps (HID and fluorescent) it became possible to increase growth to rates close to that in summer months. Supplementary lighting is used during low light periods of the year and anytime to ensure consistent total daily irradiance for research plants.« less

In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters.

PubMed

Hama, Kotaro; Provost, Elayne; Baranowski, Timothy C; Rubinstein, Amy L; Anderson, Jennifer L; Leach, Steven D; Farber, Steven A

2009-02-01

Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.

In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters

PubMed Central

Hama, Kotaro; Provost, Elayne; Baranowski, Timothy C.; Rubinstein, Amy L.; Anderson, Jennifer L.; Leach, Steven D.; Farber, Steven A.

2009-01-01

Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae. PMID:19056761

DNA strand-displacement-induced fluorescence enhancement for highly sensitive and selective assay of multiple microRNA in cancer cells.

PubMed

Wu, Ping; Tu, Yunqiu; Qian, Yingdan; Zhang, Hui; Cai, Chenxin

2014-01-28

We report a new strategy for evaluating multiple miRNA expressions in cancer cells based on DNA strand-displacement-induced fluorescence enhancement. This assay has the ability to discriminate the target from even single-base mismatched sequences or other miRNAs.

Highly Efficient Multiple-Anchored Fluorescent Probe for the Detection of Aniline Vapor Based on Synergistic Effect: Chemical Reaction and PET.

PubMed

Jiao, Zinuo; Zhang, Yu; Xu, Wei; Zhang, Xiangtao; Jiang, Haibo; Wu, Pengcheng; Fu, Yanyan; He, Qingguo; Cao, Huimin; Cheng, Jiangong

2017-05-26

A multiple-anchored fluorescent probe ((((hexane-1,6-diylbis(2,7-bis(4-formyl)-phenyl)-9H-fluorine-9,9-diyl))-bis(hexane-6,1-diyl))-bis(9H-carbazole-9,3,6-triyl))-tetrakis(benzene-4,1-diyl))-tetraformyl-(8FP-2F) with eight aldehyde groups was designed and synthesized. The molecule has four branches and highly twisted structure. Furthermore, it tends to self-assemble into nanospheres, which is beneficial for gaseous analyte penetration and high fluorescence quantum efficiency. Among gaseous analytes, detection of aniline vapor is extraordinarily important in the control of environmental issues and human diseases. Herein, 8FP-2F was introduced to detect aniline vapor with distinguished sensitivity and selectivity via simple Schiff base reaction at room temperature. After exposure to saturate aniline vapor, the 89% fluorescence of 8FP-2F was quenched in 50 s and the detection limit was as low as 3 ppb. Further study showed the suitable HOMO/LUMO energy levels and matched orbital symmetry between probe and aniline molecules ensured chemical reaction and PET process work together. The synergistic effect resulted in a significant sensing performance and fluorescence quenching toward aniline vapor. Moreover, the multiple active sites structure of 8FP-2F means it could be applied for constructing many interesting structures and highly efficient organic optoelectronic functional materials.

Indocyanine green fluorescence angiography for quantitative evaluation of in situ parathyroid gland perfusion and function after total thyroidectomy.

PubMed

Lang, Brian Hung-Hin; Wong, Carlos K H; Hung, Hing Tsun; Wong, Kai Pun; Mak, Ka Lun; Au, Kin Bun

2017-01-01

Because the fluorescent light intensity on an indocyanine green fluorescence angiography reflects the blood perfusion within a focused area, the fluorescent light intensity in the remaining in situ parathyroid glands may predict postoperative hypocalcemia risk after total thyroidectomy. Seventy patients underwent intraoperative indocyanine green fluorescence angiography after total thyroidectomy. Any parathyroid glands with a vascular pedicle was left in situ while any parathyroid glands without pedicle or inadvertently removed was autotransplanted. After total thyroidectomy, an intravenous 2.5 mg indocyanine green fluorescence angiography was given and real-time fluorescent images of the thyroid bed were recorded using the SPY imaging system (Novadaq, Ontario, Canada). The fluorescent light intensity of each indocyanine green fluorescence angiography as well as the average and greatest fluorescent light intensity in each patient were calculated. Postoperative hypocalcemia was defined as adjusted calcium <2.00 mmol/L within 24 hours. The fluorescent light intensity between discolored and normal-looking indocyanine green fluorescence angiographies was similar (P = .479). No patients with a greatest fluorescent light intensity >150% developed postoperative hypocalcemia while 9 (81.8%) patients with a greatest fluorescent light intensity ≤150% did. Similarly, no patients with an average fluorescent light intensity >109% developed PH while 9 (30%) with an average fluorescent light intensity ≤109% did. The greatest fluorescent light intensity was more predictive than day-0 postoperative hypocalcemia (P = .027) and % PTH drop day-0 to 1 (P < .001). Indocyanine green fluorescence angiography is a promising operative adjunct in determining residual parathyroid glands function and predicting postoperative hypocalcemia risk after total thyroidectomy. Copyright © 2016 Elsevier Inc. All rights reserved.

Fluorescence Approaches to Growing Macromolecule Crystals

NASA Technical Reports Server (NTRS)

Pusey, Marc; Forsythe, Elizabeth; Achari, Aniruddha

2006-01-01

Trace fluorescent labeling, typically < 1%, can be a powerful aid in macromolecule crystallization. Precipitation concentrates a solute, and crystals are the most densely packed solid form. The more densely packed the fluorescing material, the more brightly the emission from it, and thus fluorescence intensity of a solid phase is a good indication of whether one has crystals or not. The more brightly fluorescing crystalline phase is easily distinguishable, even when embedded in an amorphous precipitate. This approach conveys several distinct advantages: one can see what the protein is doing in response to the imposed conditions, and distinguishing between amorphous and microcrystalline precipitated phases are considerably simpler. The higher fluorescence intensity of the crystalline phase led us to test if we could derive crystallization conditions from screen outcomes which had no obvious crystalline material, but simply "bright spots" in the precipitated phase. Preliminary results show that the presence of these bright spots, not observable under white light, is indeed a good indicator of potential crystallization conditions.

Side-entry laser-beam zigzag irradiation of multiple channels in a microchip for simultaneous and highly sensitive detection of fluorescent analytes.

PubMed

Anazawa, Takashi; Yokoi, Takahide; Uchiho, Yuichi

2015-09-01

A simple and highly sensitive technique for laser-induced fluorescence detection on multiple channels in a plastic microchip was developed, and its effectiveness was demonstrated by laser-beam ray-trace simulations and experiments. In the microchip, with refractive index nC, A channels and B channels are arrayed alternately and respectively filled with materials with refractive indexes nA for electrophoresis analysis and nB for laser-beam control. It was shown that a laser beam entering from the side of the channel array traveled straight and irradiated all A channels simultaneously and effectively because the refractive actions by the A and B channels were counterbalanced according to the condition nA < nC < nB. This technique is thus called "side-entry laser-beam zigzag irradiation". As a demonstration of the technique, when nC = 1.53, nA = 1.41, nB = 1.66, and the cross sections of both eight A channels and seven B channels were the same isosceles trapezoids with 97° base angle, laser-beam irradiation efficiency on the eight A channels by the simulations was 89% on average and coefficient of variation was 4.4%. These results are far superior to those achieved by other conventional methods such as laser-beam expansion and scanning. Furthermore, fluorescence intensity on the eight A channels determined by the experiments agreed well with that determined by the simulations. Therefore, highly sensitive and uniform fluorescence detection on eight A channels was achieved. It is also possible to fabricate the microchips at low cost by plastic-injection molding and to make a simple and compact detection system, thereby promoting actual use of the proposed side-entry laser-beam zigzag irradiation in various fields.

Ambiguous dependence of fluorescence intensity of trees on chlorophyll concentration

NASA Astrophysics Data System (ADS)

Zavoruev, Valeriy V.; Zavorueva, Elena N.

2014-11-01

Using fluorimetry Junior PAM (Heinz Walz GmbH, Germany) fluorescence parameters of leaves Prinsepia sinensis, Crataegus chlorocarca M, Acer negúndo, Bétula péndula are studied. It was found that the dependence of maximum fluorescence (Fm) plants on the concentration of chlorophyll depends on the sampling method during of vegetation. The correctness of sampling proves during vegetation is substantiated.

A fluorescence spectroscopy study of traditional Chinese medicine Angelica

NASA Astrophysics Data System (ADS)

Zhao, Hongyan; Song, Feng; Liu, Shujing; Chen, Guiyang; Wei, Chen; Liu, Yanling; Liu, Jiadong

2013-10-01

By measuring the fluorescence spectra of Chinese medicine (CM) Angelica water solutions with different concentrations from 0.025 to 2.5 mg/mL, results showed that the fluorescence intensity was proportional to the concentration. Through fluorescence spectra of Angelica solution under different pH values, results indicated coumarin compounds were the active ingredients of Angelica. We also observed fluorescence quenching of the Angelica solution in the presence of spherical silver nanoparticles with radius of 12 nm. Keeping a certain value for the volume of the silver nanoparticles, the fluorescence intensity at 402 nm was linearly proportional to the Angelica in the range of 1-3 mg/mL.

Fluorescent image tracking velocimeter

DOEpatents

Shaffer, Franklin D.

1994-01-01

A multiple-exposure fluorescent image tracking velocimeter (FITV) detects and measures the motion (trajectory, direction and velocity) of small particles close to light scattering surfaces. The small particles may follow the motion of a carrier medium such as a liquid, gas or multi-phase mixture, allowing the motion of the carrier medium to be observed, measured and recorded. The main components of the FITV include: (1) fluorescent particles; (2) a pulsed fluorescent excitation laser source; (3) an imaging camera; and (4) an image analyzer. FITV uses fluorescing particles excited by visible laser light to enhance particle image detectability near light scattering surfaces. The excitation laser light is filtered out before reaching the imaging camera allowing the fluoresced wavelengths emitted by the particles to be detected and recorded by the camera. FITV employs multiple exposures of a single camera image by pulsing the excitation laser light for producing a series of images of each particle along its trajectory. The time-lapsed image may be used to determine trajectory and velocity and the exposures may be coded to derive directional information.

Apparatus and method for measuring fluorescence intensities at a plurality of wavelengths and lifetimes

DOEpatents

Buican, T.N.

1993-05-04

Apparatus and method is described for measuring intensities at a plurality of wavelengths and lifetimes. A source of multiple-wavelength electromagnetic radiation is passed through a first interferometer modulated at a first frequency, the output thereof being directed into a sample to be investigated. The light emitted from the sample as a result of the interaction thereof with the excitation radiation is directed into a second interferometer modulated at a second frequency, and the output detected and analyzed. In this manner excitation, emission, and lifetime information may be obtained for a multiplicity of fluorochromes in the sample.

Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells.

PubMed

Gurskaya, N G; Staroverov, D B; Lukyanov, K A

2016-01-01

Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines. © 2016 Elsevier Inc. All rights reserved.

Intensive (Daily) Behavior Therapy for School Refusal: A Multiple Baseline Case Series

ERIC Educational Resources Information Center

Tolin, David F.; Whiting, Sara; Maltby, Nicholas; Diefenbach, Gretchen J.; Lothstein, Mary Anne; Hardcastle, Surrey; Catalano, Amy; Gray, Krista

2009-01-01

The following multiple baseline case series examines school refusal behavior in 4 male adolescents. School refusal symptom presentation was ascertained utilizing a functional analysis from the School Refusal Assessment Scale (Kearney, 2002). For the majority of cases, treatment was conducted within a 15-session intensive format over a 3-week…

Light propagation analysis for fluorescence measurements of a molecular probe in the brain

NASA Astrophysics Data System (ADS)

Asai, Kota; Togashi, Takuya; Okada, Eiji

2017-04-01

Light propagation in the slab head model that consists of five types of tissues was calculated to estimate the fluorescent intensity emerged from a molecular probe in the brain by a Monte Carlo simulation. The thickness of the scalp, skull and cerebrospinal fluid layer was varied to analyze the influence of the thickness of the superficial tissues on the fluorescent intensity detected on the scalp surface. The fluorescent intensity is exponentially reduced with increasing the depth of the brain surface. The thickness of the cerebrospinal fluid layer more significantly affects the fluorescent intensity than that of the scalp and skull.

Fluorescence detection system for microfluidic droplets

NASA Astrophysics Data System (ADS)

Chen, Binyu; Han, Xiaoming; Su, Zhen; Liu, Quanjun

2018-05-01

In microfluidic detection technology, because of the universality of optical methods in laboratory, optical detection is an attractive solution for microfluidic chip laboratory equipment. In addition, the equipment with high stability and low cost can be realized by integrating appropriate optical detection technology on the chip. This paper reports a detection system for microfluidic droplets. Photomultiplier tubes (PMT) is used as a detection device to improve the sensitivity of detection. This system improves the signal to noise ratio by software filtering and spatial filter. The fluorescence intensity is proportional to the concentration of the fluorescence and intensity of the laser. The fluorescence micro droplets of different concentrations can be distinguished by this system.

Fluorescence from a single Symbiodinium cell

NASA Astrophysics Data System (ADS)

Guzman, Christine; Han, Xue; Shoguchi, Eiichi; Chormaic, Síle Nic

2018-07-01

The partnership between coral and its algal symbionts, Symbiodinium, is crucial to the global environment. Yet, the regulatory process within the photosynthetic machinery of Symbiodinium is still not clearly understood. Here, we studied the influence of light stress from focussed red and blue lasers on single Symbiodinium cells. Fluorescence signals were measured to show cell response. Increasing the incident laser power or the exposure time resulted in an increase followed by a decline in fluorescence intensity. The trend of fluorescence intensity changes was associated with mechanisms of light use efficiency, non-photochemical quenching, photoinhibition, and repair of the cell. Our study provides new approaches to studying the photobiology and physiology of Symbiodinium cells.

Multiple high-intensity focused ultrasound probes for kidney-tissue ablation.

PubMed

Häcker, Axel; Chauhan, Sunita; Peters, Kristina; Hildenbrand, Ralf; Marlinghaus, Ernst; Alken, Peter; Michel, Maurice Stephan

2005-10-01

To investigate kidney-tissue ablation by high-intensity focused ultrasound (HIFU) using multiple and single probes. Ultrasound beams (1.75 MHz) produced by a piezoceramic element (focal distance 80 mm) were focused at the center of renal parenchyma. One of the three probes (mounted on a jig) could also be used for comparison with a single probe at comparable power ratings. Lesion dimensions were examined in perfused and unperfused ex vivo porcine kidneys at different power levels (40, 60, and 80 W) and treatment times (4, 6, and 8 seconds). At identical power levels, the lesions induced by multiple probes were larger than those induced by a single probe. Lesion size increased with increasing pulse duration and generator power. The sizes and shapes of the lesions were predictably repeatable in all samples. Lesions in perfused kidneys were smaller than those in unperfused kidneys. Ex vivo, kidney-tissue ablation by means of multiple HIFU probes offers significant advantages over single HIFU probes in respect of lesion size and formation. These advantages need to be confirmed by tests in vivo at higher energy levels.

Quantifying fluorescence enhancement for slowly diffusing single molecules in plasmonic near fields

NASA Astrophysics Data System (ADS)

Caldarola, Martín; Pradhan, Biswajit; Orrit, Michel

2018-03-01

Gold nanorods are extensively used for single-molecule fluorescence enhancement as they are easy to synthesize, bio-compatible, and provide high light confinement at their nanometer-sized tips. The current way to estimate fluorescence enhancement relies on binned time traces or on fluorescence correlation spectroscopy. We report on novel ways to extract the enhancement factor in a single-molecule enhancement experiment, avoiding the arbitrary selection of one or a few high-intensity burst(s). These new estimates for the enhancement factor make use of the whole distribution of intensity bursts or of the interphoton delay distribution, which avoids the arbitrary binning of the fluorescence intensity time traces. We present experimental results on the bi-dimensional case, experimentally achieved using a lipid bilayer to support the diffusion of fluorophores. We support our findings with histograms of fluorescence bursts and with an analytical derivation of the interphoton delay distribution of (nearly) immobilized emitters from the fluorescence intensity profile.

Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching.

PubMed

Iijima, Issei; Hohsaka, Takahiro

2009-04-17

Position-specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. We have developed a method for the incorporation of visible-wavelength-fluorescent non-natural amino acids into proteins in a cell-free translation system. Using this technique, we introduced one or two BODIPY-linked amino acids into maltose-binding protein (MBP) to obtain MBP derivatives showing ligand-dependent changes in fluorescence intensity or intensity ratio. BODIPY-FL-aminophenylalanine was incorporated in place of 15 tyrosines, as well as the N-terminal Lys1, and the C-terminal Lys370 of MBP. Fluorescence measurements revealed that MBP containing a BODIPY-FL moiety in place of Tyr210 showed a 13-fold increase in fluorescence upon binding of maltose. Tryptophan-to-phenylalanine substitutions suggest that the increase in fluorescence was the result of a decrease in the quenching of BODIPY-FL by tryptophan located around the binding site. MBP containing a BODIPY-558 moiety also showed a maltose-dependent increase in fluorescence. BODIPY-FL was then additionally incorporated in place of Lys1 of the BODIPY-558-containing MBP as a response to the amber codon. Fluorescence measurements with excitation of BODIPY-FL showed a large change in fluorescence intensity ratio (0.13 to 1.25) upon binding of maltose; this change can be attributed to fluorescence resonance energy transfer (FRET) and maltose-dependent quenching of BODIPY-558. These results demonstrate the usefulness of the position-specific incorporation of fluorescent amino acids in the fluorescence-based detection of protein functions.

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

PubMed

Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

2017-05-15

5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

Improved Charge-Transfer Fluorescent Dyes

NASA Technical Reports Server (NTRS)

Meador, Michael

2005-01-01

Improved charge-transfer fluorescent dyes have been developed for use as molecular probes. These dyes are based on benzofuran nuclei with attached phenyl groups substituted with, variously, electron donors, electron acceptors, or combinations of donors and acceptors. Optionally, these dyes could be incorporated as parts of polymer backbones or as pendant groups or attached to certain surfaces via self-assembly-based methods. These dyes exhibit high fluorescence quantum yields -- ranging from 0.2 to 0.98, depending upon solvents and chemical structures. The wavelengths, quantum yields, intensities, and lifetimes of the fluorescence emitted by these dyes vary with (and, hence, can be used as indicators of) the polarities of solvents in which they are dissolved: In solvents of increasing polarity, fluorescence spectra shift to longer wavelengths, fluorescence quantum yields decrease, and fluorescence lifetimes increase. The wavelengths, quantum yields, intensities, and lifetimes are also expected to be sensitive to viscosities and/or glass-transition temperatures. Some chemical species -- especially amines, amino acids, and metal ions -- quench the fluorescence of these dyes, with consequent reductions in intensities, quantum yields, and lifetimes. As a result, the dyes can be used to detect these species. Another useful characteristic of these dyes is a capability for both two-photon and one-photon absorption. Typically, these dyes absorb single photons in the ultraviolet region of the spectrum (wavelengths < 400 nm) and emit photons in the long-wavelength ultraviolet, visible, and, when dissolved in some solvents, near-infrared regions. In addition, these dyes can be excited by two-photon absorption at near-infrared wavelengths (600 to 800 nm) to produce fluorescence spectra identical to those obtained in response to excitation by single photons at half the corresponding wavelengths (300 to 400 nm). While many prior fluorescent dyes exhibit high quantum yields

Fluorescence lifetime measurements in flow cytometry

NASA Astrophysics Data System (ADS)

Beisker, Wolfgang; Klocke, Axel

1997-05-01

Fluorescence lifetime measurements provide insights int eh dynamic and structural properties of dyes and their micro- environment. The implementation of fluorescence lifetime measurements in flow cytometric systems allows to monitor large cell and particle populations with high statistical significance. In our system, a modulated laser beam is used for excitation and the phase shift of the fluorescence signal recorded with a fast computer controlled digital oscilloscope is processed digitally to determine the phase shift with respect to a reference beam by fast fourier transform. Total fluorescence intensity as well as other parameters can be determined simultaneously from the same fluorescence signal. We use the epi-illumination design to allow the use of high numerical apertures to collect as much light as possible to ensure detection of even weak fluorescence. Data storage and processing is done comparable to slit-scan flow cytometric data using data analysis system. The results are stored, displayed, combined with other parameters and analyzed as normal listmode data. In our report we discuss carefully the signal to noise ratio for analog and digital processed lifetime signals to evaluate the theoretical minimum fluorescence intensity for lifetime measurements. Applications to be presented include DNA staining, parameters of cell functions as well as different applications in non-mammalian cells such as algae.

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

PubMed

Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.

PubMed

Burnette, Dylan T; Sengupta, Prabuddha; Dai, Yuhai; Lippincott-Schwartz, Jennifer; Kachar, Bechara

2011-12-27

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

Site-specific multipoint fluorescence measurement system with end-capped optical fibers.

PubMed

Song, Woosub; Moon, Sucbei; Lee, Byoung-Cheol; Park, Chul-Seung; Kim, Dug Young; Kwon, Hyuk Sang

2011-07-10

We present the development and implementation of a spatially and spectrally resolved multipoint fluorescence correlation spectroscopy (FCS) system utilizing multiple end-capped optical fibers and an inexpensive laser source. Specially prepared end-capped optical fibers placed in an image plane were used to both collect fluorescence signals from the sample and to deliver signals to the detectors. The placement of independently selected optical fibers on the image plane was done by monitoring the end-capped fiber tips at the focus using a CCD, and fluorescence from specific positions of a sample were collected by an end-capped fiber, which could accurately represent light intensities or spectral data without incurring any disturbance. A fast multipoint spectroscopy system with a time resolution of ∼1.5 ms was then implemented using a prism and an electron multiplying charge coupled device with a pixel binning for the region of interest. The accuracy of our proposed system was subsequently confirmed by experimental results, based on an FCS analysis of microspheres in distilled water. We expect that the proposed multipoint site-specific fluorescence measurement system can be used as an inexpensive fluorescence measurement tool to study many intracellular and molecular dynamics in cell biology. © 2011 Optical Society of America

Imaging a Large Sample with Selective Plane Illumination Microscopy Based on Multiple Fluorescent Microsphere Tracking

NASA Astrophysics Data System (ADS)

Ryu, Inkeon; Kim, Daekeun

2018-04-01

A typical selective plane illumination microscopy (SPIM) image size is basically limited by the field of view, which is a characteristic of the objective lens. If an image larger than the imaging area of the sample is to be obtained, image stitching, which combines step-scanned images into a single panoramic image, is required. However, accurately registering the step-scanned images is very difficult because the SPIM system uses a customized sample mount where uncertainties for the translational and the rotational motions exist. In this paper, an image registration technique based on multiple fluorescent microsphere tracking is proposed in the view of quantifying the constellations and measuring the distances between at least two fluorescent microspheres embedded in the sample. Image stitching results are demonstrated for optically cleared large tissue with various staining methods. Compensation for the effect of the sample rotation that occurs during the translational motion in the sample mount is also discussed.

Morphological spot counting from stacked images for automated analysis of gene copy numbers by fluorescence in situ hybridization.

PubMed

Grigoryan, Artyom M; Dougherty, Edward R; Kononen, Juha; Bubendorf, Lukas; Hostetter, Galen; Kallioniemi, Olli

2002-01-01

Fluorescence in situ hybridization (FISH) is a molecular diagnostic technique in which a fluorescent labeled probe hybridizes to a target nucleotide sequence of deoxyribose nucleic acid. Upon excitation, each chromosome containing the target sequence produces a fluorescent signal (spot). Because fluorescent spot counting is tedious and often subjective, automated digital algorithms to count spots are desirable. New technology provides a stack of images on multiple focal planes throughout a tissue sample. Multiple-focal-plane imaging helps overcome the biases and imprecision inherent in single-focal-plane methods. This paper proposes an algorithm for global spot counting in stacked three-dimensional slice FISH images without the necessity of nuclei segmentation. It is designed to work in complex backgrounds, when there are agglomerated nuclei, and in the presence of illumination gradients. It is based on the morphological top-hat transform, which locates intensity spikes on irregular backgrounds. After finding signals in the slice images, the algorithm groups these together to form three-dimensional spots. Filters are employed to separate legitimate spots from fluorescent noise. The algorithm is set in a comprehensive toolbox that provides visualization and analytic facilities. It includes simulation software that allows examination of algorithm performance for various image and algorithm parameter settings, including signal size, signal density, and the number of slices.

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

PubMed

Rich, Ryan M; Stankowska, Dorota L; Maliwal, Badri P; Sørensen, Thomas Just; Laursen, Bo W; Krishnamoorthy, Raghu R; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal

2013-02-01

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

PubMed Central

Rich, Ryan M.; Stankowska, Dorota L.; Maliwal, Badri P.; Sørensen, Thomas Just; Laursen, Bo W.; Krishnamoorthy, Raghu R.; Gryczynski, Zygmunt; Borejdo, Julian

2013-01-01

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background. PMID:23254457

A fluorescence detection of D-penicillamine based on Cu(2+)-induced fluorescence quenching system of protein-stabilized gold nanoclusters.

PubMed

Wang, Peng; Li, Bang Lin; Li, Nian Bing; Luo, Hong Qun

2015-01-25

In this contribution, a luminescent gold nanoclusters which were synthesized by bovine serum albumin as novel fluorescent probes were successfully utilized for the determination of D-penicillamine for the first time. Cupric ion was employed to quench the strong fluorescence of the gold nanoclusters, whereas the addition of D-penicillamine caused obvious restoration of fluorescence intensity of the Cu(2+)-gold nanoclusters system. Under optimum conditions, the increment in fluorescence intensity of Cu(2+)-gold nanoclusters system caused by D-penicillamine was linearly proportional to the concentration of D-penicillamine in the range of 2.0×10(-5)-2.39×10(-4) M. The detection limit for D-penicillamine was 5.4×10(-6) M. With the off-on fluorescence signal at 650 nm approaching the near-infrared region, the present sensor for D-penicillamine detection had high sensitivity and low spectral interference. Furthermore, the novel gold nanoclusters-based fluorescent sensor has been applied to the determination of D-penicillamine in real biological samples with satisfactory results. Copyright © 2014 Elsevier B.V. All rights reserved.

Helix-coil transition of a four-way DNA junction observed by multiple fluorescence parameters.

PubMed

Vámosi, György; Clegg, Robert M

2008-10-16

The thermal denaturation of immobile four-way DNA ("Holliday-") junctions with 17 base pair arms was studied via fluorescence spectroscopic measurements. Two arms of the molecule were labeled at the 5'-end with fluorescein and tetramethylrhodamine, respectively. Melting was monitored by the fluorescence intensity of the dyes, the fluorescence anisotropy of tetramethylrhodamine, and Forster resonance energy transfer (FRET) between fluorescein and rhodamine. To fit the thermal denaturation curves of the four-way junctions, two basic thermodynamic models were tested: (1) all-or-none transitions assuming a molecularity of one, two, or four and (2) a statistical "zipper" model. The all-or-none models correspond to reaction mechanisms assuming that the cooperative melting unit (that is, the structure changing from complete helix to complete coil) consists of (1) one arm, (2) two neighboring arms (which have one continuous strand common to the two arms), or (3) all four arms. In each case, the melting of the cooperative unit takes place in a single step. The tetramolecular reaction model (four-arm melting) yielded unrealistically low van't Hoff enthalpy and entropy values, whereas the monomolecular model (one-arm melting) resulted in a poor fit to the experimental data. The all-or-none bimolecular (two neighboring arm model) fit gave intermediate standard enthalpy change (Delta H) values between those expected for the melting of a duplex with a total length between the helix lengths of one and two arms (17 and 34 base pairs). Simulations according to the zipper model fit the experimental curves best when the length of the simulated duplex was assumed to be 34 base pairs, the length of a single strand. This suggests that the most important parameter determining the melting behavior of the molecule is the end-to-end distance of the strands (34 bases) rather than the length of the individual arms (17 base pairs) and that the equilibrium concentration of partially denatured

IR-stimulated visible fluorescence in pink and brown diamond.

PubMed

Byrne, K S; Chapman, J G; Luiten, A N

2014-03-19

Irradiation of natural pink and brown diamond by middle-ultraviolet light (photon energy ϵ ≥ 4.1 eV ) is seen to induce anomalous fluorescence phenomena at N3 defect centres (structure N3-V). When diamonds primed in this fashion are subsequently exposed to infrared light (even with a delay of many hours), a transient burst of blue N3 fluorescence is observed. The dependence of this IR-triggered fluorescence on pump wavelength and intensity suggest that this fluorescence phenomena is intrinsically related to pink diamond photochromism. An energy transfer process between N3 defects and other defect species can account for both the UV-induced fluorescence intensity changes, and the apparent optical upconversion of IR light. From this standpoint, we consider the implications of this N3 fluorescence behaviour for the current understanding of pink diamond photochromism kinetics.

Conditions for NIR fluorescence-guided tumor resectioning in preclinical lung cancer model (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kim, Minji; Quan, Yuhua; Choi, Byeong Hyun; Choi, Yeonho; Kim, Hyun Koo; Kim, Beop-Min

2016-03-01

Pulmonary nodule could be identified by intraoperative fluorescence imaging system from systemic injection of indocyanine green (ICG) which achieves enhanced permeability and retention (EPR) effects. This study was performed to evaluate optimal injection time of ICG for detecting cancer during surgery in rabbit lung cancer model. VX2 carcinoma cell was injected in rabbit lung under fluoroscopic computed tomography-guidance. Solitary lung cancer was confirmed on positron emitting tomography with CT (PET/CT) 2 weeks after inoculation. ICG was administered intravenously and fluorescent intensity of lung tumor was measured using the custom-built intraoperative color and fluorescence merged imaging system (ICFIS) for 15 hours. Solitary lung cancer was resected through thoracoscopic version of ICFIS. ICG was observed in all animals. Because Lung has fast blood pulmonary circulation, Fluorescent signal showed maximum intensity earlier than previous studies in other organs. Fluorescent intensity showed maximum intensity within 6-9 hours in rabbit lung cancer. Overall, Fluorescent intensity decreased with increasing time, however, all tumors were detectable using fluorescent images until 12 hours. In conclusion, while there had been studies in other organs showed that optimal injection time was at least 24 hours before operation, this study showed shorter optimal injection time at lung cancer. Since fluorescent signal showed the maximum intensity within 6-9 hours, cancer resection could be performed during this time. This data informed us that optimal injection time of ICG should be evaluated in each different solid organ tumor for fluorescent image guided surgery.

Determination of ethambutol by a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Wu, Wen-Ying; Yang, Ji-Yuan; Du, Li-Ming; Wu, Hao; Li, Chang-Feng

2011-08-01

The competitive reaction between ethambutol and two fluorescent probes (i.e., berberine and palmatine) for occupancy of the cucurbit[7]uril (CB[7]) cavity was studied by spectrofluorometry. The CB[7] reacts with these probes to form stable complexes, and the fluorescence intensity of the complexes is greatly enhanced. In addition, the excitation and emission wavelengths of their complexes moved to wavelengths of 343 nm and 495 nm, respectively. However, the addition of ethambutol dramatically quenches the fluorescence intensity of the two complexes. Accordingly, a couple of new fluorescence quenching methods for the determination of ethambutol were established. The methods can be applied for quantifying ethambutol. A linear relationship between the fluorescence quenching values (Δ F) and ethambutol concentration exists in the range of 5.0-1000.0 ng mL -1, with a correlation coefficient ( r) of 0.9997. The detection limit is 1.7 ng mL -1. The fluorescent probe of berberine has higher sensitivity than palmatine. This paper also discusses the mechanism of fluorescence indicator probes.

Novel and remarkable enhanced-fluorescence system based on gold nanoclusters for detection of tetracycline.

PubMed

Yang, Xiaoming; Zhu, Shanshan; Dou, Yao; Zhuo, Yan; Luo, Yawen; Feng, Yuanjiao

2014-05-01

Tetracycline and Eu(3+), while coexisting, usually appear as a complex by chelating. This complex shows low fluorescence intensity, leading to its limitation of analytical goals. Gold nanoclusters (AuNCs), emerging as novel nano-material, are attracting increasing attentions in multiple fields. Herein, gold nanoclusters first function as a fluorescence-enhanced reagent rather than a conventional fluorescent-probe, and a dramatic enhanced-fluorescence system was built based on Eu(3+)-Tetracycline complex (EuTC) by introducing gold nanoclusters. Simultaneously, three types of gold nanoclusters were employed for exploring various conditions likely affecting the system, which demonstrate that no other gold nanoclusters than DNA-templated gold nanoclusters enormously caused fluorescence-enhancement of EuTC. Moreover, this enhanced-fluorescence system permitted available detection of tetracycline (TC) in a linear range of 0.01-5 μM, with a detection limit of 4 nM at a signal-to-noise ratio of 3. Significantly, the practicality of this method for detection of TC in human urine and milk samples was validated, demonstrating its advantages of simplicity, sensitivity and low cost. Interestingly, this system described here is probably promising for kinds of applications based on its dramatically enhanced-fluorescence. © 2013 Published by Elsevier B.V.

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Chih Wei; Schwab, Mark; Hill, Steven C.

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300–350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm 2, dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ~500–700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increasedmore » to 1 kW/cm 2 or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ~ 650–800 nm with 633-nm CW laser excitation. In conclusion, we attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.« less

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles.

PubMed

Lai, Chih Wei; Schwab, Mark; Hill, Steven C; Santarpia, Joshua; Pan, Yong-Le

2016-05-30

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300-350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm², dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ∼500-700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increased to 1 kW/cm² or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ∼ 650-800 nm with 633-nm CW laser excitation. We attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles

DOE PAGES

Lai, Chih Wei; Schwab, Mark; Hill, Steven C.; ...

2016-05-19

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300–350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm 2, dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ~500–700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increasedmore » to 1 kW/cm 2 or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ~ 650–800 nm with 633-nm CW laser excitation. In conclusion, we attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.« less

Metal-enhanced fluorescence of single green fluorescent protein (GFP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu Yi; Zhang Jian; Lakowicz, Joseph R.

2008-11-28

The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein-protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. In this report, we observed the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and decreases the decay time. Single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, we observed longer durationmore » time and suppressed blinking. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure.« less

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

Reconstruction From Multiple Particles for 3D Isotropic Resolution in Fluorescence Microscopy.

PubMed

Fortun, Denis; Guichard, Paul; Hamel, Virginie; Sorzano, Carlos Oscar S; Banterle, Niccolo; Gonczy, Pierre; Unser, Michael

2018-05-01

The imaging of proteins within macromolecular complexes has been limited by the low axial resolution of optical microscopes. To overcome this problem, we propose a novel computational reconstruction method that yields isotropic resolution in fluorescence imaging. The guiding principle is to reconstruct a single volume from the observations of multiple rotated particles. Our new operational framework detects particles, estimates their orientation, and reconstructs the final volume. The main challenge comes from the absence of initial template and a priori knowledge about the orientations. We formulate the estimation as a blind inverse problem, and propose a block-coordinate stochastic approach to solve the associated non-convex optimization problem. The reconstruction is performed jointly in multiple channels. We demonstrate that our method is able to reconstruct volumes with 3D isotropic resolution on simulated data. We also perform isotropic reconstructions from real experimental data of doubly labeled purified human centrioles. Our approach revealed the precise localization of the centriolar protein Cep63 around the centriole microtubule barrel. Overall, our method offers new perspectives for applications in biology that require the isotropic mapping of proteins within macromolecular assemblies.

Ns-scaled time-gated fluorescence lifetime imaging for forensic document examination

NASA Astrophysics Data System (ADS)

Zhong, Xin; Wang, Xinwei; Zhou, Yan

2018-01-01

A method of ns-scaled time-gated fluorescence lifetime imaging (TFLI) is proposed to distinguish different fluorescent substances in forensic document examination. Compared with Video Spectral Comparator (VSC) which can examine fluorescence intensity images only, TFLI can detect questioned documents like falsification or alteration. TFLI system can enhance weak signal by accumulation method. The two fluorescence intensity images of the interval delay time tg are acquired by ICCD and fitted into fluorescence lifetime image. The lifetimes of fluorescence substances are represented by different colors, which make it easy to detect the fluorescent substances and the sequence of handwritings. It proves that TFLI is a powerful tool for forensic document examination. Furthermore, the advantages of TFLI system are ns-scaled precision preservation and powerful capture capability.

Filter Enhances Fluorescent-Penetrant-Inspecting Borescope

NASA Technical Reports Server (NTRS)

Molina, Orlando G.

1990-01-01

Slip-on eyepiece for commercial ultraviolet-light borescope reduces both amount of short-wave ultraviolet light that reaches viewer's eye and apparent intensity of unwanted reflections of white light from surfaces undergoing inspection. Fits on stock eyepiece of borescope, which illuminates surface inspected with intense ultraviolet light. Surface, which is treated with fluorescent dye, emits bright-green visible light wherever dye penetrates - in cracks and voids. Eyepiece contains deep-yellow Wratten 15 (G) filter, which attenuates unwanted light strongly but passes yellow-green fluorescence so defects seen clearly.

A label-free, fluorescence based assay for microarray

NASA Astrophysics Data System (ADS)

Niu, Sanjun

intensity of excitation light. The fluorescence contrast is used to quantify the amount of probe-target hybridization. A mathematical model that considers multiple reflections and scattering is developed to explain the mechanism of the fluorescence contrast which depends on the thickness of the PS film. Scattering is the dominant factor that contributes to the contrast. The potential of this assay to detect single nucleotide polymorphism is also tested.

Fabrication of Indocyanine Green and 2H, 3H-perfluoropentane loaded microbubbles for fluorescence and ultrasound imaging

NASA Astrophysics Data System (ADS)

He, Yutong; Wu, Qiang; Ma, Rong; Chang, Shufang; Shao, Pengfei; Xu, Ronald

2016-03-01

As a near-infrared (NIR) fluorescence dye, Indocyanine Green (ICG) has not gained broader clinical applications, owing to its multiple limitations such as concentration-dependent aggregation, low fluorescence quantum yield, poor physicochemical stability and rapid elimination from the body. In the meanwhile, 2H,3H-perfluoropentane (H-PFP) has been widely studied in ultrasound imaging as a vehicle for targeted delivery of contrast agents and drugs. We synthesized a novel dual-modal fluorescence and ultrasound contrast agent by encapsulating ICG and H-PFP in lipid microbubbles using a liquid-driven coaxial flow focusing (LDCFF) process. Uniform microbubbles with the sizes ranging from 1-10um and great ICG loading efficiency was achieved by this method. Our benchtop experiments showed that ICG/H-PFP microbubbles exhibited less aggregation, increased fluorescence intensity and more stable photostability compared to free ICG aqueous solution. Our phantom experiments demonstrated that ICG/H-PFP microbubbles enhanced the imaging contrasts in fluorescence imaging and ultrasonography. Our animal experiments indicated that ICG/H-PFP microbubbles extended the ICG life time and facilitated dual mode fluorescence and ultrasound imaging in vivo.

Enhanced speed in fluorescence imaging using beat frequency multiplexing

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke

2016-03-01

Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

Laser-excited fluorescence for measuring atmospheric pollution

NASA Technical Reports Server (NTRS)

Menzies, R. T.

1975-01-01

System measures amount of given pollutant at specific location. Infrared laser aimed at location has wavelength that will cause molecules of pollutant to fluoresce. Detector separates fluorescence from other radiation and measures its intensity to indicate concentration of pollutant.

Angular shaping of fluorescence from synthetic opal-based photonic crystal.

PubMed

Boiko, Vitalii; Dovbeshko, Galyna; Dolgov, Leonid; Kiisk, Valter; Sildos, Ilmo; Loot, Ardi; Gorelik, Vladimir

2015-01-01

Spectral, angular, and temporal distributions of fluorescence as well as specular reflection were investigated for silica-based artificial opals. Periodic arrangement of nanosized silica globules in the opal causes a specific dip in the defect-related fluorescence spectra and a peak in the reflectance spectrum. The spectral position of the dip coincides with the photonic stop band. The latter is dependent on the size of silica globules and the angle of observation. The spectral shape and intensity of defect-related fluorescence can be controlled by variation of detection angle. Fluorescence intensity increases up to two times at the edges of the spectral dip. Partial photobleaching of fluorescence was observed. Photonic origin of the observed effects is discussed.

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

PubMed Central

Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

2011-01-01

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

Fluorescence Stability of Mercaptopropionic Acid Capped Cadmium Telluride Quantum Dots in Various Biochemical Buffers.

PubMed

Borse, Vivek; Kashikar, Adisha; Srivastava, Rohit

2018-04-01

Quantum dots are the semiconductor nanocrystals having unique optical and electronic properties. Quantum dots are category of fluorescent labels utilized for biological tagging, biosensing, bioassays, bioimaging and in vivo imaging as they exhibit very small size, signal brightness, photostability, tuning of light emission range, longer photoluminescence decay time as compared to organic dyes. In this work, we have synthesized and characterized mercaptopropionic acid capped cadmium telluride quantum dots (MPA-CdTe QDs) using hydrothermal method. The study further reports fluorescence intensity stability of quantum dots suspended in different buffers of varying concentration (1-100 mM), stored at various photophysical conditions. Fluorescence intensity values were reduced with increase in buffer concentration. When the samples were stored at room temperature in ambient light condition the quantum dots suspended in different buffers lost the fluorescence intensity after day 15 (except TRIS II). Fluorescence intensity values were found stable for more than 30 days when the samples were stored in dark condition. Samples stored in refrigerator displayed modest fluorescence intensity even after 300 days of storage. Thus, storage of MPA-CdTe QDs in refrigerator may be the suitable choice to maintain its fluorescence stability for longer time for further application.

Red fluorescent biofilm: the thick, the old, and the cariogenic

PubMed Central

Volgenant, Catherine M.C.; Hoogenkamp, Michel A.; Buijs, Mark J.; Zaura, Egija; ten Cate, Jacob (Bob) M.; van der Veen, Monique H.

2016-01-01

Background Some dental plaque fluoresces red. The factors involved in this fluorescence are yet unknown. Objective The aim of this study was to assess systematically the effect of age, thickness, and cariogenicity on the extent of red fluorescence produced by in vitro microcosm biofilms. Design The effects of biofilm age and thickness on red fluorescence were tested in a constant depth film fermentor (CDFF) by growing biofilms of variable thicknesses that received a constant supply of defined mucin medium (DMM) and eight pulses of sucrose/day. The influence of cariogenicity on red fluorescence was tested by growing biofilm on dentin disks receiving DMM, supplemented with three or eight pulses of sucrose/day. The biofilms were analyzed at different time points after inoculation, up to 24 days. Emission spectra were measured using a fluorescence spectrophotometer (λexc405 nm) and the biofilms were photographed with a fluorescence camera. The composition of the biofilms was assessed using 454-pyrosequecing of the 16S rDNA gene. Results From day 7 onward, the biofilms emitted increasing intensities of red fluorescence as evidenced by the combined red fluorescence peaks. The red fluorescence intensity correlated with biofilm thickness but not in a linear way. Biofilm fluorescence also correlated with the imposed cariogenicity, evidenced by the induced dentin mineral loss. Increasing the biofilm age or increasing the sucrose pulsing frequency led to a shift in the microbial composition. These shifts in composition were accompanied by an increase in red fluorescence. Conclusions The current study shows that a thicker, older, or more cariogenic biofilm results in a higher intensity of red fluorescence. PMID:27060056

Adaptive thresholding image series from fluorescence confocal scanning laser microscope using orientation intensity profiles

NASA Astrophysics Data System (ADS)

Feng, Judy J.; Ip, Horace H.; Cheng, Shuk H.

2004-05-01

Many grey-level thresholding methods based on histogram or other statistic information about the interest image such as maximum entropy and so on have been proposed in the past. However, most methods based on statistic analysis of the images concerned little about the characteristics of morphology of interest objects, which sometimes could provide very important indication which can help to find the optimum threshold, especially for those organisms which have special texture morphologies such as vasculature, neuro-network etc. in medical imaging. In this paper, we propose a novel method for thresholding the fluorescent vasculature image series recorded from Confocal Scanning Laser Microscope. After extracting the basic orientation of the slice of vessels inside a sub-region partitioned from the images, we analysis the intensity profiles perpendicular to the vessel orientation to get the reasonable initial threshold for each region. Then the threshold values of those regions near the interest one both in x-y and optical directions have been referenced to get the final result of thresholds of the region, which makes the whole stack of images look more continuous. The resulting images are characterized by suppressing both noise and non-interest tissues conglutinated to vessels, while improving the vessel connectivities and edge definitions. The value of the method for idealized thresholding the fluorescence images of biological objects is demonstrated by a comparison of the results of 3D vascular reconstruction.

Detection of bacterial infection of agave plants by laser-induced fluorescence

NASA Astrophysics Data System (ADS)

Cervantes-Martinez, Jesus; Flores-Hernandez, Ricardo; Rodriguez-Garay, Benjamin; Santacruz-Ruvalcaba, Fernando

2002-05-01

Greenhouse-grown plants of Agave tequilana Weber var. azul were inoculated with Erwinia carotovora, the causal agent of stem soft rot. We investigated the laser-induced fluorescence (LIF) of agave plants to determine whether LIF can be used as a noninvasive sensing tool for pathological studies. The LIF technique was also investigated as a means of detecting the effect of the polyamine biosynthesis inhibitor beta-hydroxyethylhydrazine as a bactericide against the pathogenic bacterium Erwinia carotovora. A He-Ne laser at 632.8 nm was used as the excitation source, and in vivo fluorescence emission spectra were recorded in the 660-790-range. Fluorescence maxima were at 690 and 740 nm. The infected plants that were untreated with the bactericide showed a definite increase in fluorescence intensity at both maxima within the first three days after infection. Beginning on the fifth day, a steady decrease in fluorescence intensity was observed, with a greater effect at 740 than at 690 nm. After 30 days there was no fluorescence. The infected plants that had been treated with the bactericide showed no significant change in fluorescence compared with that of the uninfected plants. The ratio of fluorescence intensities was determined to be F 690 nm/F 740 nm for all treatments. These studies indicate that LIF measurements of agave plants may be used for the early detection of certain types of disease and for determining the effect of a bactericide on bacteria. The results also showed that fluorescence intensity ratios can be used as a reliable indicator of the progress of disease.

Detection of bacterial infection of agave plants by laser-induced fluorescence.

PubMed

Cervantes-Martínez, Jesús; Flores-Hernández, Ricardo; Rodríguez-Garay, Benjamin; Santacruz-Ruvalcaba, Fernando

2002-05-01

Greenhouse-grown plants of Agave tequilana Weber var. azul were inoculated with Erwinia carotovora, the causal agent of stem soft rot. We investigated the laser-induced fluorescence (LIF) of agave plants to determine whether LIF can be used as a noninvasive sensing tool for pathological studies. The LIF technique was also investigated as a means of detecting the effect of the polyamine biosynthesis inhibitor beta-hydroxyethylhydrazine as a bactericide against the pathogenic bacterium Erwinia carotovora. A He-Ne laser at 632.8 nm was used as the excitation source, and in vivo fluorescence emission spectra were recorded in the 660-790-range. Fluorescence maxima were at 690 and 740 nm. The infected plants that were untreated with the bactericide showed a definite increase in fluorescence intensity at both maxima within the first three days after infection. Beginning on the fifth day, a steady decrease in fluorescence intensity was observed, with a greater effect at 740 than at 690 nm. After 30 days there was no fluorescence. The infected plants that had been treated with the bactericide showed no significant change in fluorescence compared with that of the uninfected plants. The ratio of fluorescence intensities was determined to be F 690 nm/F 740 nm for all treatments. These studies indicate that LIF measurements of agave plants may be used for the early detection of certain types of disease and for determining the effect of a bactericide on bacteria. The results also showed that fluorescence intensity ratios can be used as a reliable indicator of the progress of disease.

Application of novel low-intensity nonscanning fluorescence lifetime imaging microscopy for monitoring excited state dynamics in individual chloroplasts and living cells of photosynthetic organisms

NASA Astrophysics Data System (ADS)

Eckert, Hann-Jörg; Petrášek, Zdeněk; Kemnitz, Klaus

2006-10-01

Picosecond fluorescence lifetime imaging microscopy (FLIM) provides a most valuable tool to analyze the primary processes of photosynthesis in individual cells and chloroplasts of living cells. In order to obtain correct lifetimes of the excited states, the peak intensity of the exciting laser pulses as well as the average intensity has to be sufficiently low to avoid distortions of the kinetics by processes such as singlet-singlet annihilation, closing of the reaction centers or photoinhibition. In the present study this requirement is achieved by non-scanning wide-field FLIM based on time- and space-correlated single-photon counting (TSCSPC) using a novel microchannel plate photomultiplier with quadrant anode (QA-MCP) that allows parallel acquisition of time-resolved images under minimally invasive low-excitation conditions. The potential of the wide-field TCSPC method is demonstrated by presenting results obtained from measurements of the fluorescence dynamics in individual chloroplasts of moss leaves and living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.

Quantitative, spectrally-resolved intraoperative fluorescence imaging

PubMed Central

Valdés, Pablo A.; Leblond, Frederic; Jacobs, Valerie L.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.

2012-01-01

Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field. PMID:23152935

Tuning Fluorescence Direction with Plasmonic Metal–Dielectric– Metal Substrates

PubMed Central

Choudhury, Sharmistha Dutta; Badugu, Ramachandram; Nowaczyk, Kazimierz; Ray, Krishanu; Lakowicz, Joseph R.

2013-01-01

Controlling the emission properties of fluorophores is essential for improving the performance of fluorescence-based techniques in modern biochemical research, medical diagnosis, and sensing. Fluorescence emission is isotropic in nature, which makes it difficult to capture more than a small fraction of the total emission. Metal– dielectric–metal (MDM) substrates, discussed in this Letter, convert isotropic fluorescence into beaming emission normal to the substrate. This improves fluorescence collection efficiency and also opens up new avenues for a wide range of fluorescence-based applications. We suggest that MDM substrates can be readily adapted for multiple uses, such as in microarray formats, for directional fluorescence studies of multiple probes or for molecule-specific sensing with a high degree of spatial control over the fluorescence emission. SECTION: Physical Processes in Nanomaterials and Nanostructures PMID:24013521

Fuel Mix Impacts from Transportation Fuel Carbon Intensity Standards in Multiple Jurisdictions

NASA Astrophysics Data System (ADS)

Witcover, J.

2017-12-01

Fuel carbon intensity standards have emerged as an important policy in jurisdictions looking to target transportation greenhouse gas (GHG) emissions for reduction. A carbon intensity standard rates transportation fuels based on analysis of lifecycle GHG emissions, and uses a system of deficits and tradable, bankable credits to reward increased use of fuels with lower carbon intensity ratings while disincentivizing use of fuels with higher carbon intensity ratings such as conventional fossil fuels. Jurisdictions with carbon intensity standards now in effect include California, Oregon, and British Columbia, all requiring 10% reductions in carbon intensity of the transport fuel pool over a 10-year period. The states and province have committed to grow demand for low carbon fuels in the region as part of collaboration on climate change policies. Canada is developing a carbon intensity standard with broader coverage, for fuels used in transport, industry, and buildings. This study shows a changing fuel mix in affected jurisdictions under the policy in terms of shifting contribution of transportation energy from alternative fuels and trends in shares of particular fuel pathways. It contrasts program designs across the jurisdictions with the policy, highlights the opportunities and challenges these pose for the alternative fuel market, and discusses the impact of having multiple policies alongside federal renewable fuel standards and sometimes local carbon pricing regimes. The results show how the market has responded thus far to a policy that incentivizes carbon saving anywhere along the supply chain at lowest cost, in ways that diverged from a priori policy expectations. Lessons for the policies moving forward are discussed.

Neoplasm diagnostics based on fluorescence of polymethine dyes

NASA Astrophysics Data System (ADS)

Samtsov, Michael P.; Voropay, Eugene S.; Chalov, Vadim N.; Zhavrid, Edvard A.

2002-05-01

Investigated polymethine dye TICS has near IR bands of fluorescence and absorption within the transparency region of biological tissues. It can be detected up to 1.5 cm from the surface of the skin. The intensity of a fluorescence signal of TICS is linear for doses up to 2 mg/kg in both tumor and muscle tissue. The ratio of an intensity of light induced fluorescence in tumor tissue to one in muscle tissue is up to 3.6 for rapidly growing tumors. The retention time of TICS is 7 days in all tissues. TICS can be used in the detection of tumor boundaries and tumor internal structure.

Motor Oil Classification Based on Time-Resolved Fluorescence

PubMed Central

Mu, Taotao; Chen, Siying; Zhang, Yinchao; Guo, Pan; Chen, He; Meng, Fandong

2014-01-01

A time-resolved fluorescence (TRF) technique is presented for classifying motor oils. The system is constructed with a third harmonic Nd:YAG laser, a spectrometer, and an intensified charge coupled device (ICCD) camera. Steady-state and time-resolved fluorescence (TRF) measurements are reported for several motor oils. It is found that steady-state fluorescence is insufficient to distinguish the motor oil samples. Then contour diagrams of TRF intensities (CDTRFIs) are acquired to serve as unique fingerprints to identify motor oils by using the distinct TRF of motor oils. CDTRFIs are preferable to steady-state fluorescence spectra for classifying different motor oils, making CDTRFIs a particularly choice for the development of fluorescence-based methods for the discrimination and characterization of motor oils. The two-dimensional fluorescence contour diagrams contain more information, not only the changing shapes of the LIF spectra but also the relative intensity. The results indicate that motor oils can be differentiated based on the new proposed method, which provides reliable methods for analyzing and classifying motor oils. PMID:24988439

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675.

PubMed

Konold, Patrick E; Yoon, Eunjin; Lee, Junghwa; Allen, Samantha L; Chapagain, Prem P; Gerstman, Bernard S; Regmi, Chola K; Piatkevich, Kiryl D; Verkhusha, Vladislav V; Joo, Taiha; Jimenez, Ralph

2016-08-04

Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.

Multiple Velocity Profile Measurements in Hypersonic Flows Using Sequentially-Imaged Fluorescence Tagging

NASA Technical Reports Server (NTRS)

Bathel, Brett F.; Danehy, Paul M.; Inman, Jennifer A.; Jones, Stephen B.; Ivey,Christopher b.; Goyne, Christopher P.

2010-01-01

Nitric-oxide planar laser-induced fluorescence (NO PLIF) was used to perform velocity measurements in hypersonic flows by generating multiple tagged lines which fluoresce as they convect downstream. For each laser pulse, a single interline, progressive scan intensified CCD (charge-coupled device) camera was used to obtain two sequential images of the NO molecules that had been tagged by the laser. The CCD configuration allowed for sub-microsecond acquisition of both images, resulting in sub-microsecond temporal resolution as well as sub-mm spatial resolution (0.5-mm horizontal, 0.7-mm vertical). Determination of axial velocity was made by application of a cross-correlation analysis of the horizontal shift of individual tagged lines. A numerical study of measured velocity error due to a uniform and linearly-varying collisional rate distribution was performed. Quantification of systematic errors, the contribution of gating/exposure duration errors, and the influence of collision rate on temporal uncertainty were made. Quantification of the spatial uncertainty depended upon the signal-to-noise ratio of the acquired profiles. This velocity measurement technique has been demonstrated for two hypersonic flow experiments: (1) a reaction control system (RCS) jet on an Orion Crew Exploration Vehicle (CEV) wind tunnel model and (2) a 10-degree half-angle wedge containing a 2-mm tall, 4-mm wide cylindrical boundary layer trip. The experiments were performed at the NASA Langley Research Center's 31-Inch Mach 10 Air Tunnel.

Fluorescence and fluorescence-lifetime imaging microscopy (FLIM) to characterize yeast strains by autofluorescence

NASA Astrophysics Data System (ADS)

Bhatta, H.; Goldys, E. M.; Ma, J.

2006-02-01

We characterised populations of wild type baking and brewing yeast cells using intrinsic fluorescence and fluorescence lifetime microscopy, in order to obtain quantitative identifiers of different strains. The cell autofluorescence was excited at 405 nm and observed within 440-540 nm range where strong cell to cell variability was observed. The images were analyzed using customised public domain software, which provided information on cell size, intensity and texture-related features. In light of significant diversity of the data, statistical methods were utilized to assess the validity of the proposed quantitative identifiers for strain differentiation. The Kolmogorov-Smirnov test was applied to confirm that empirical distribution functions for size, intensity and entropy for different strains were statistically different. These characteristics were followed with culture age of 24, 48 and 72 h, (the latter corresponding to a stationary growth phase) and size, and to some extent entropy, were found to be independent of age. The fluorescence intensity presented a distinctive evolution with age, different for each of the examined strains. The lifetime analysis revealed a short decay time component of 1.4 ns and a second, longer one with the average value of 3.5 ns and a broad distribution. High variability of lifetime values within cells was observed however a lifetime texture feature in the studied strains was statistically different.

Laser-saturated fluorescence measurements in laminar sooting diffusion flames

NASA Technical Reports Server (NTRS)

Wey, Changlie

1993-01-01

The hydroxyl radical is known to be one of the most important intermediate species in the combustion processes. The hydroxyl radical has also been considered a dominant oxidizer of soot particles in flames. In this investigation the hydroxyl concentration profiles in sooting diffusion flames were measured by the laser-saturated fluorescence (LSF) method. The temperature distributions in the flames were measured by the two-line LSF technique and by thermocouple. In the sooting region the OH fluorescence was too weak to make accurate temperature measurements. The hydroxyl fluorescence profiles for all four flames presented herein show that the OH fluorescence intensities peaked near the flame front. The OH fluorescence intensity dropped sharply toward the dark region of the flame and continued declining to the sooting region. The OH fluorescence profiles also indicate that the OH fluorescence decreased with increasing height in the flames for all flames investigated. Varying the oxidizer composition resulted in a corresponding variation in the maximum OH concentration and the flame temperature. Furthermore, it appears that the maximum OH concentration for each flame increased with increasing flame temperature.

Theoretical investigation for excitation light and fluorescence signal of fiber optical sensor using tapered fiber tip.

PubMed

Yuan, Yinquan; Ding, Liyun

2011-10-24

For fiber optical sensor made of tapered fiber tip, the effects of the geometrical parameters of tapered tip on two important factors have been investigated. One factor is the intensity of the evanescent wave into fluorescent layer through core-medium interface; the other is the intensity of fluorescence signal transmitted from fluorescent layer to measurement end. A dependence relation of the intensity of fluorescence signal transmitted from fluorescent layer to measurement end upon the geometrical parameters of tapered tip has been obtained. Theoretical results show that the intensity of the evanescent wave into fluorescent layer rises with the decrease of the end diameter of tapered tip, and the increase of the tip length; and the transmitted power of fluorescence signal increases linearly with the increase of the tip length due to the contribution of the side area of tapered tip. © 2011 Optical Society of America

Experimental assessment of fluorescence microscopy signal enhancement by stimulated emission

NASA Astrophysics Data System (ADS)

Dake, Fumihiro; Yazawa, Hiroki

2017-10-01

The quantity of photons generated during fluorescence microscopy is principally determined by the quantum yield of the fluorescence dyes and the optical power of the excitation beam. However, even though low quantum yields can produce poor images, it is challenging to tune this parameter, while increasing the power of the excitation beam often results in photodamage. Here, we propose the use of stimulated emission (SE) as a means of enhancing both the signal intensity and signal-to-noise ratio during confocal fluorescence microscopy. This work experimentally confirmed that both these factors can be enhanced by SE radiation, through generating a greater number of photons than are associated with the standard fluorescence signal. We also propose the concept of stimulated emission enhancing fluorescence (SEEF) microscopy, which employs both the SE and fluorescence signals, and demonstrate that the intensity of an SEEF signal is greater than those of the individual SE and fluorescence signals.

Fluorescence correlation spectroscopy: principles and applications.

PubMed

Bacia, Kirsten; Haustein, Elke; Schwille, Petra

2014-07-01

Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed. © 2014 Cold Spring Harbor Laboratory Press.

Conjugated polyelectrolyte based real-time fluorescence assay for phospholipase C.

PubMed

Liu, Yan; Ogawa, Katsu; Schanze, Kirk S

2008-01-01

A fluorescence turnoff assay for phospholipase C (PLC) from Clostridium perfringens is developed based on the reversible interaction between the natural substrate, phosphatidylcholine, and a fluorescent, water-soluble conjugated polyelectrolyte (CPE). The fluorescence intensity of the CPE in water is increased substantially by the addition of the phospholipid due to the formation of a CPE-lipid complex. Incubation of the CPE-lipid complex with the enzyme PLC causes the fluorescence intensity to decrease (turnoff sensor); the response arises due to PLC-catalyzed hydrolysis of the phosphatidylcholine, which effectively disrupts the CPE-lipid complex. The PLC assay operates with phospholipid substrate concentrations in the micromolar range, and the analytical detection limit for PLC is <1 nM. The optimized assay provides a convenient, rapid, and real-time sensor for PLC activity. The real-time fluorescence intensity from the CPE can be converted to substrate concentration by using an ex situ calibration curve, allowing PLC-catalyzed reaction rates and kinetic parameters to be determined. PLC activation by Ca2+ and inhibition by EDTA and fluoride ion are demonstrated using the optimized sensor.

Time-resolved fluorescence and FCS studies of dye-doped DNA

NASA Astrophysics Data System (ADS)

Nicolaou, N.; Marsh, R. J.; Blacker, T.; Armoogum, D. A.; Bain, A. J.

2009-08-01

Fluorescence lifetime, anisotropy and intensity dependent single molecule fluorescence correlation spectroscopy (I-FCS) are used to investigate the mechanism of fluorescence saturation in a free and nucleotide bound fluorophore (NR6104) in an antioxidising ascorbate buffer. Nucleotide attachment does not appreciably affect the fluorescence lifetime of the probe and there is a decrease in the rate of intersystem crossing relative to that of triplet state deactivation. The triplet state fraction is seen to plateau at 72% (G-attached) and 80% (free fluorophore) in agreement with these observations. Measurements of translational diffusion times show no intensity dependence for excitation intensities between 1 and 105kW cm-2 and photobleaching is therefore negligible. The dominant mechanism of fluorescence saturation is thus triplet state formation. I-FCS measurements for Rhodamine 6G in water were compared with those in the ascorbate buffer. In water the triplet fraction was saturated at considerably higher powers (45% at ca. 1.5 × 103kW cm-2) than in the ascorbate buffer (55%ca. 1 1kW cm-2)

Fluorescence detection of dental calculus

NASA Astrophysics Data System (ADS)

Gonchukov, S.; Biryukova, T.; Sukhinina, A.; Vdovin, Yu

2010-11-01

This work is devoted to the optimization of fluorescence dental calculus diagnostics in optical spectrum. The optimal wavelengths for fluorescence excitation and registration are determined. Two spectral ranges 620 - 645 nm and 340 - 370 nm are the most convenient for supra- and subgingival calculus determination. The simple implementation of differential method free from the necessity of spectrometer using was investigated. Calculus detection reliability in the case of simple implementation is higher than in the case of spectra analysis at optimal wavelengths. The use of modulated excitation light and narrowband detection of informative signal allows us to decrease essentially its diagnostic intensity even in comparison with intensity of the low level laser dental therapy.

Application of fluorescence spectroscopy and imaging in the detection of a photosensitizer in photodynamic therapy

NASA Astrophysics Data System (ADS)

Zang, Lixin; Zhao, Huimin; Zhang, Zhiguo; Cao, Wenwu

2017-02-01

Photodynamic therapy (PDT) is currently an advanced optical technology in medical applications. However, the application of PDT is limited by the detection of photosensitizers. This work focuses on the application of fluorescence spectroscopy and imaging in the detection of an effective photosenzitizer, hematoporphyrin monomethyl ether (HMME). Optical properties of HMME were measured and analyzed based on its absorption and fluorescence spectra. The production mechanism of its fluorescence emission was analyzed. The detection device for HMME based on fluorescence spectroscopy was designed. Ratiometric method was applied to eliminate the influence of intensity change of excitation sources, fluctuates of excitation sources and photo detectors, and background emissions. The detection limit of this device is 6 μg/L, and it was successfully applied to the diagnosis of the metabolism of HMME in the esophageal cancer cells. To overcome the limitation of the point measurement using fluorescence spectroscopy, a two-dimensional (2D) fluorescence imaging system was established. The algorithm of the 2D fluorescence imaging system is deduced according to the fluorescence ratiometric method using bandpass filters. The method of multiple pixel point addition (MPPA) was used to eliminate fluctuates of signals. Using the method of MPPA, SNR was improved by about 30 times. The detection limit of this imaging system is 1.9 μg/L. Our systems can be used in the detection of porphyrins to improve the PDT effect.

In vivo assessment of wound re-epithelialization by UV fluorescence excitation imaging

NASA Astrophysics Data System (ADS)

Wang, Ying; Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Williams, Maura; Farinelli, William; Anderson, R. R.; Franco, Walfre

2017-02-01

Background and Objectives: We have previously demonstrated the efficacy of a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds in vitro. This system can image highly-proliferating cellular processes (295/340 nm excitation/emission wavelengths) to study epithelialization in a cultured wound model. The objective of the current work is to evaluate the suitability of u-FEI for monitoring wound re-epithelialization in vivo. Study Design: Full-thickness wounds were created in the tail of rats and imaged weekly using u-FEI at 295/340nm excitation/emission wavelengths. Histology was used to investigate the correlation between the spatial distribution and intensity of fluorescence and the extent of wound epithelialization. In addition, the expression of the nuclear protein Ki67 was used to confirm the association between the proliferation of keratinocyte cells and the intensity of fluorescence. Results: Keratinocytes forming neo-epidermis exhibited higher fluorescence intensity than the keratinocytes not involved in re-epithelialization. In full-thickness wounds the fluorescence first appeared at the wound edge where keratinocytes initiated the epithelialization process. Fluorescence intensity increased towards the center as the keratinocytes partially covered the wound. As the wound healed, fluorescence decreased at the edges and was present only at the center as the keratinocytes completely covered the wound at day 21. Histology demonstrated that changes in fluorescence intensity from the 295/340nm band corresponded to newly formed epidermis. Conclusions: u-FEI at 295/340nm allows visualization of proliferating keratinocyte cells during re-epithelialization of wounds in vivo, potentially providing a quantitative, objective and simple method for evaluating wound closure in the clinic.

Synthesis and fluorescence properties of some difluoroboron β-diketonate complexes and composite containing PMMA

NASA Astrophysics Data System (ADS)

Xing, Dongye; Hou, Yanjun; Niu, Haijun

2018-03-01

A series of difluoroboron β-diketonate complexes, containing the indon-β-diketonate ligand carrying methyl or methoxyl substituents was synthesized. The crystal structures of the complexes were confirmed by single crystal X-ray diffraction studies. The fluorescence properties of compounds were studied in solution state, solid state and on PMMA polymer matrix. The photophysical data of compounds 2a-2d exhibited strong fluorescence and photostability under the ultraviolet light (Hg lamp). The complex 2b showed higher fluorescence intensity in solution state as compared to other complexes of the series. The complexes 2c and 2d showed higher fluorescence intensity in the solid state, which are ascribed to the stronger π-π interactions between ligands in the solid state. The introduction of methoxyl or methyl groups on the benzene rings enhanced the absorption intensity, emission intensity, quantum yields and fluorescence lifetimes due to their electron-donating nature. Furthermore, the complex 2b was doped into the PMMA to produce hybrid materials, where the PMMA matrix acted as sensitizer for the central boron ion to enhance the fluorescence emission intensity and quantum yields.

Three-dimensional fluorescence analysis of chernozem humic acids and their electrophoretic fractions

NASA Astrophysics Data System (ADS)

Trubetskoi, O. A.; Trubetskaya, O. E.

2017-09-01

Polyacrylamide gel electrophoresis in combination with size-exclusion chromatography (SEC-PAGE) has been used to obtain stable electrophoretic fractions of different molecular size (MS) from chernozem humic acids (HAs). Three-dimensional fluorescence charts of chernozem HAs and their fractions have been obtained for the first time, and all fluorescence excitation-emission maxima have been identified in the excitation wavelength range of 250-500 nm. It has been found that fractionation by the SEC-PAGE method results in a nonuniform distribution of protein- and humin-like fluorescence of the original HA preparation among the electrophoretic fractions. The electrophoretic fractions of the highest and medium MSs have only the main protein-like fluorescence maximum and traces of humin-like fluorescence. In the electrophoretic fraction of the lowest MS, the intensity of protein-like fluorescence is low, but the major part of humin-like fluorescence is localized there. Relationships between the intensity of protein-like fluorescence and the weight distribution of amino acids have been revealed, as well as between the degree of aromaticity and the intensity of humin-like fluorescence in electrophoretic fractions of different MSs. The obtained relationships can be useful in the interpretation of the spatial structural organization and ecological functions of soil HAs.

Real-time quantitative fluorescence measurement of microscale cell culture analog systems

NASA Astrophysics Data System (ADS)

Oh, Taek-il; Kim, Donghyun; Tatosian, Daniel; Sung, Jong Hwan; Shuler, Michael

2007-02-01

A microscale cell culture analog (μCCA) is a cell-based lab-on-a-chip assay that, as an animal surrogate, is applied to pharmacological studies for toxicology tests. A μCCA typically comprises multiple chambers and microfluidics that connect the chambers, which represent animal organs and blood flow to mimic animal metabolism more realistically. A μCCA is expected to provide a tool for high-throughput drug discovery. Previously, a portable fluorescence detection system was investigated for a single μCCA device in real-time. In this study, we present a fluorescence-based imaging system that provides quantitative real-time data of the metabolic interactions in μCCAs with an emphasis on measuring multiple μCCA samples simultaneously for high-throughput screening. The detection system is based on discrete optics components, with a high-power LED and a charge-coupled device (CCD) camera as a light source and a detector, for monitoring cellular status on the chambers of each μCCA sample. Multiple samples are characterized mechanically on a motorized linear stage, which is fully-automated. Each μCCA sample has four chambers, where cell lines MES-SA/DX- 5, and MES-SA (tumor cells of human uterus) have been cultured. All cell-lines have been transfected to express the fusion protein H2B-GFP, which is a human histone protein fused at the amino terminus to EGFP. As a model cytotoxic drug, 10 μM doxorubicin (DOX) was used. Real-time quantitative data of the intensity loss of enhanced green fluorescent protein (EGFP) during cell death of target cells have been collected over several minutes to 40 hours. Design issues and improvements are also discussed.

Nine New Fluorescent Probes

NASA Astrophysics Data System (ADS)

Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

1989-06-01

Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model.

PubMed

Wang, Sibo; Yang, Tao; Zhang, Xuyong; Xia, Jie; Guo, Jun; Wang, Xiaoyi; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

2016-06-01

Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation.

Epi-Fluorescence Microscopy

PubMed Central

Webb, Donna J.; Brown, Claire M.

2012-01-01

Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new “hard” coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes that are the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented. PMID:23026996

Locally adaptive MR intensity models and MRF-based segmentation of multiple sclerosis lesions

NASA Astrophysics Data System (ADS)

Galimzianova, Alfiia; Lesjak, Žiga; Likar, Boštjan; Pernuš, Franjo; Špiclin, Žiga

2015-03-01

Neuroimaging biomarkers are an important paraclinical tool used to characterize a number of neurological diseases, however, their extraction requires accurate and reliable segmentation of normal and pathological brain structures. For MR images of healthy brains the intensity models of normal-appearing brain tissue (NABT) in combination with Markov random field (MRF) models are known to give reliable and smooth NABT segmentation. However, the presence of pathology, MR intensity bias and natural tissue-dependent intensity variability altogether represent difficult challenges for a reliable estimation of NABT intensity model based on MR images. In this paper, we propose a novel method for segmentation of normal and pathological structures in brain MR images of multiple sclerosis (MS) patients that is based on locally-adaptive NABT model, a robust method for the estimation of model parameters and a MRF-based segmentation framework. Experiments on multi-sequence brain MR images of 27 MS patients show that, compared to whole-brain model and compared to the widely used Expectation-Maximization Segmentation (EMS) method, the locally-adaptive NABT model increases the accuracy of MS lesion segmentation.

Direct spectrometry: a new alternative for measuring the fluorescence of composite resins and dental tissues.

PubMed

da Silva, Tm; de Oliveira, Hpm; Severino, D; Balducci, I; Huhtala, Mfrl; Gonçalves, Sep

2014-01-01

The aim of this study was to evaluate the fluorescence intensity of different composite resins and compare those values with the fluorescence intensity of dental tissues. Different composite resins were used to make 10 discs (2 mm in depth and 4 mm in diameter) of each brand, divided into groups: 1) Z (Filtek Z350, 3M ESPE), 2) ES (Esthet-X, Dentsply), 3) A (Amelogen Plus, Ultradent), 4) DVS (Durafill-VS, Heraeus Kulzer) with 2 mm composite resin for enamel (A2), 5) OES ([Esthet-X] opaque-OA [1 mm] + enamel-A2 [1 mm]); 6) ODVSI ([Charisma-Opal/Durafill-VSI], opaque-OM (1 mm) + translucent [1mm]), and 7) DVSI ([Durafill- VSI] translucent [2 mm]). Dental tissue specimens were obtained from human anterior teeth cut in a mesiodistal direction to obtain enamel, dentin, and enamel/dentin samples (2 mm). The fluorescence intensity of specimens was directly measured using an optic fiber associated with a spectrometer (Ocean Optics USB 4000) and recorded in graphic form (Origin 8.0 program). Data were submitted to statistical analysis using Dunnet, Tukey, and Kruskall-Wallis tests. Light absorption of the composite resins was obtained in a spectral range from 250 to 450 nm, and that of dental tissues was between 250 and 300 nm. All composite resins were excited at 398 nm and exhibited maximum emissions of around 485 nm. Fluorescence intensity values for all of the resins showed statistically significant differences (measured in arbitrary units [AUs]), with the exception of groups Z and DVS. Group DVSI had the highest fluorescence intensity values (13539 AU), followed by ODVS (10440 AU), DVS (10146 AU), ES (3946 AU), OES (3841 AU), A (3540 AU), and Z (1146 AU). The fluorescence intensity values for the composite resins differed statistically from those of dental tissues (E=1380 AU; D=6262 AU; E/D=3251 AU). The opacity interfered with fluorescence intensity, and group Z demonstrated fluorescence intensity values closest to that of tooth enamel. It is concluded that the

Direct fluorescence characterisation of a picosecond seeded optical parametric amplifier

NASA Astrophysics Data System (ADS)

Stuart, N. H.; Bigourd, D.; Hill, R. W.; Robinson, T. S.; Mecseki, K.; Patankar, S.; New, G. H. C.; Smith, R. A.

2015-02-01

The temporal intensity contrast of high-power lasers based on optical parametric amplification (OPA) can be limited by parametric fluorescence from the non-linear gain stages. Here we present a spectroscopic method for direct measurement of unwanted parametric fluorescence widely applicable from unseeded to fully seeded and saturated OPA operation. Our technique employs simultaneous spectroscopy of fluorescence photons slightly outside the seed bandwidth and strongly attenuated light at the seed central wavelength. To demonstrate its applicability we have characterised the performance of a two-stage picosecond OPA pre-amplifier with 2.8×105 gain, delivering 335 μJ pulses at 1054 nm. We show that fluorescence from a strongly seeded OPA is reduced by ~500× from the undepleted to full pump depletion regimes. We also determine the vacuum fluctuation driven noise term seeding this OPA fluorescence to be 0.7±0.4 photons ps-1 nm-1 bandwidth. The resulting shot-to-shot statistics highlights a 1.5% probability of a five-fold and 0.3% probability of a ten-fold increase of fluorescence above the average value. Finally, we show that OPA fluorescence can be limited to a few-ps pedestal with 3×10-9 temporal intensity contrast 1.3 ps ahead of an intense laser pulse, a level highly attractive for large scale chirped-pulse OPA laser systems.

Time-resolved multicolor two-photon excitation fluorescence microscopy of cells and tissues

NASA Astrophysics Data System (ADS)

Zheng, Wei

2014-11-01

Multilabeling which maps the distribution of different targets is an indispensable technique in many biochemical and biophysical studies. Two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with conventional fluorescence labeling techniques such as genetically encoded fluorescent protein (FP) and fluorescent dyes staining could be a powerful tool for imaging living cells. However, the challenge is that the excitation and emission wavelength of these endogenous fluorophores and fluorescent labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores, fluorescent proteins and fluorescent dyes were excited in their optimal wavelengths simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and wavelength domains. Cellular organelles such as nucleus, mitochondria, microtubule and endoplasmic reticulum, were clearly revealed in the TPEF images. The simultaneous imaging of multiple fluorophores of cells will greatly aid the study of sub-cellular compartments and protein localization.

Time-resolved fluorescence spectroscopic study of flavin fluorescence in purified enzymes of bioluminescent bacteria

NASA Astrophysics Data System (ADS)

Vetrova, Elena; Kudryasheva, N.; Cheng, K.

2006-10-01

Time-resolved fluorescence intensity and anisotropy decay measurements have been used to study the environment and rotational mobility of endogenous flavin in two purified enzymes of bioluminescent bacteria, Luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri. We compared the time-resolved fluorescence parameters, intensity decay lifetimes, rotational correlation times, and their fractional contribution, of the endogeneous flavin fluorescence in each of the two enzymes in the presence or absence of quinones of different structures and redox potentials. The endogeneous flavin exhibited multi-exponential decay characteristics as compared to a single decay lifetime of around 5 ns for free flavin, suggesting a complex and heterogeneous environment of flavin bound to the enzyme. In addition, a significant increase in the rotational correlation time and a certain degree of ordering of the molecule were observed for endogenous flavin when compared to a single and fast rotational correlation time of 150 ps of free flavin. Quinone significantly altered both the lifetime and rotational characteristics of endogenous flavin suggesting specific interactions of quinones to the endogeneous flavin in the bacterial enzyme.

Electron and fluorescence spectra of a water molecule irradiated by an x-ray free-electron laser pulse

NASA Astrophysics Data System (ADS)

Schäfer, Julia M.; Inhester, Ludger; Son, Sang-Kil; Fink, Reinhold F.; Santra, Robin

2018-05-01

With the highly intense x-ray light generated by x-ray free-electron lasers (XFELs), molecular samples can be ionized many times in a single pulse. Here we report on a computational study of molecular spectroscopy at the high x-ray intensity provided by XFELs. Calculated photoelectron, Auger electron, and x-ray fluorescence spectra are presented for a single water molecule that reaches many electronic hole configurations through repeated ionization steps. The rich details shown in the spectra depend on the x-ray pulse parameters in a nonintuitive way. We discuss how the observed trends can be explained by the competition of microscopic electronic transition processes. A detailed comparison between spectra calculated within the independent-atom model and within the molecular-orbital framework highlights the chemical sensitivity of the spectral lines of multiple-hole configurations. Our results demonstrate how x-ray multiphoton ionization-related effects such as charge-rearrangement-enhanced x-ray ionization of molecules and frustrated absorption manifest themselves in the electron and fluorescence spectra.

Workflow for high-content, individual cell quantification of fluorescent markers from universal microscope data, supported by open source software.

PubMed

Stockwell, Simon R; Mittnacht, Sibylle

2014-12-16

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators. Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software(1) to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Effect of solvents on the fluorescence spectra of bacterial luciferase

NASA Astrophysics Data System (ADS)

Sukovataya, Irina E.; Tyulkova, Natalya A.; Kaykova, Elisaveta V.

2006-08-01

Bacteria luciferases catalyze the oxidation reaction of the long-chain aliphatic aldehyde and reduced flavinmononucleotide involving molecular oxygen to a respective fatty acid emitting light quanta in the visible spectrum. Fluorescence emission of luciferases from Photobacterium leiognathi dissolved in organic solvent-water mixtures was investigated. Methanol, acetone, dimethyl sulfoxide and formamide were used as organic solvents. As the methanol and acetone concentration is increased the emission maximum peak is decrease. In contrast, with dimethyl sulfoxide and formamide addition induced a increasing of the emission maximum intensity. The values of wavelength maximum (λ max) at the addition of this solvent can shows the spectra shifted to the red by about 12 nm. These increasing in the fluorescence intensity and in the λ max may be due to luciferase denaturation, resulting from the more intensive contact of chromospheres of luciferase with the solvent. At all used concentrations of methanol, acetone and formamide the shape of the fluorescence spectra was not changed. These studies demonstrate that the luciferase tryptophan fluorescence is sensitive to changes of physical-chemical property of enzyme environment. A comparison of activation/inactivation and fluorescence spectra of luciferase in methanol or acetone solutions shows that the extent of inactivation is larger than the extent of fluorescence changes at the same methanol or acetone concentration.

Research of the fluorescence detection apparatus for nutrients

NASA Astrophysics Data System (ADS)

Wang, Yu; Yan, Huimin; Ni, Xuxiang; Xu, Xiaoyi; Chen, Shibing

2015-10-01

The research of the multifunctional analyzer of Clinical Nutrition, which integrates the absorbance, luminescence, fluorescence and other optical detection methods, can overcome the functional limitations of a single technology on human nutrition analysis, and realize a rapid and accurate analysis of the nutrients. This article focuses on the design of fluorescence detection module that uses a photomultiplier tube(PMT) to detect weak fluorescence, and utilizes the single photon counting method to measure the fluorescence intensity, and then according to the relationship between the fluorescent marker and fluorescence intensity, the concentration of the analyte can be derived. Using fluorescein isothiocyanate(FITC, the most widely used fluorescein currently)to mark antibodies in the experiment, therefore, according to the maximum absorption wavelength and the maximum emission wavelength of the fluorescein isothiocyanate, to select the appropriate filters to set up the optical path. In addition, the fluorescence detection apparatus proposed in this paper uses an aspherical lens with large numerical aperture, in order to improve the capacity of signal acquisition more effectively, and the selective adoption of flexible optical fiber can realize a compact opto-mechanical structure, which is also conducive to the miniaturization of the device. The experimental results show that this apparatus has a high sensitivity, can be used for the detection and analysis of human nutrition.

The exploration of the characteristics of the hyperglycemia serum fluorescence spectrum

NASA Astrophysics Data System (ADS)

Wang, Lexin; Zhao, Zhimin; Chen, Hui; Li, Peng; Xin, Yujun

2008-12-01

Now, spectra technology is widely used in the biomedicine research,so this study investigates variation of the fluorescence spectra in different excitation wavelength, and the spectra of serum with different glucose concentration is tested in the excitation wavelength of 240nm to 280nm. The experimental result shows that the correlation between the serum fluorescence intensity and the excitation light is very close, when the excitation light is in the ultraviolet wave band, the fluorescence of serum is intensive. There is a violent fluorescence emission wavelength, which is 300nm to 410nm, while the excitation wavelength ranges from 220nm to 290nm, and the peaks wavelength are 330nm and 370nm. From 240nm to 280nm, the serum fluorescence intensity increases synchronously with the glucose concentration, and the relationship between the fluorescence peak wavelength and the glucose concentration is almost in line. In this way the blood sugar concentration can be estimated by the fluorescence spectra peak wavelength when the excitation wavelength is from 240nm to 280nm, which is effective. It provides experimental foundation for the wide use of spectra technology in medical diagnose, and the effectiv method to test the blood sugar concentration.

[Intensive care treatment of traumatic brain injury in multiple trauma patients : Decision making for complex pathophysiology].

PubMed

Trimmel, H; Herzer, G; Schöchl, H; Voelckel, W G

2017-09-01

Traumatic brain injury (TBI) and hemorrhagic shock due to uncontrolled bleeding are the major causes of death after severe trauma. Mortality rates are threefold higher in patients suffering from multiple injuries and additionally TBI. Factors known to impair outcome after TBI, namely hypotension, hypoxia, hypercapnia, acidosis, coagulopathy and hypothermia are aggravated by the extent and severity of extracerebral injuries. The mainstays of TBI intensive care may be, at least temporarily, contradictory to the trauma care concept for multiple trauma patients. In particular, achieving normotension in uncontrolled bleeding situations, maintenance of normocapnia in traumatic lung injury and thromboembolic prophylaxis are prone to discussion. Due to an ongoing uncertainty about the definition of normotensive blood pressure values, a cerebral perfusion pressure-guided cardiovascular management is of key importance. In contrast, there is no doubt that early goal directed coagulation management improves outcome in patients with TBI and multiple trauma. The timing of subsequent surgical interventions must be based on the development of TBI pathology; therefore, intensive care of multiple trauma patients with TBI requires an ongoing and close cooperation between intensivists and trauma surgeons in order to individualize patient care.

Two-photon fluorescence anisotropy imaging

NASA Astrophysics Data System (ADS)

Li, Wei; Wang, Yi; Shao, Hanrong; He, Yonghong; Ma, Hui

2006-09-01

We have developed a novel method for imaging the fluorescence intensity and anisotropy by two-photon fluorescence microscopy and tested its capability in biological application. This method is applied to model sample including FITC and FITC-CD44 antibody solution and also FITC-CD44 stained cells. The fluorescence anisotropy (FA) of FITC-CD44ab solution is higher than the FITC solution with the same concentration. The fluorescence in cell sample has even higher FA than in solution because the rotation diffusion is restrained in membrane. The method is employed to study the effect of berberine a kind of Chinese medicine, on tumor metastasis. The results indicated that tumor cell membrane fluidity is decreasing with increasing the concentration of berberine in culture medium.

Optically sectioned wide-field fluorescence lifetime imaging endoscopy enabled by structured illumination (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hinsdale, Taylor; Malik, Bilal H.; Rico-Jimenez, Jose J.; Jo, Javier A.; Maitland, Kristen C.

2016-03-01

We present a wide-field fluorescence lifetime imaging (FLIM) system with optical sectioning by structured illumination microscopy (SIM). FLIM measurements were made using a time gated ICCD camera in conjunction with a pulsed nitrogen dye laser operating at 450 nm. Intensity images were acquired at multiple time delays from a trigger initiated by a laser pulse to create a wide-field FLIM image, which was then combined with three phase SIM to provide optical sectioning. Such a mechanism has the potential to increase the reliability and accuracy of the FLIM measurements by rejecting background intensity. SIM also provides the opportunity to create volumetric FLIM images with the incorporation of scanning mechanisms for the sample plane. We present multiple embodiments of such a system: one as a free space endoscope and the other as a fiber microendoscope enabled by the introduction of a fiber bundle. Finally, we demonstrate the efficacy of such an imaging system by imaging dyes embedded in a tissue phantom.

Nanostructured Surfaces and Detection Instrumentation for Photonic Crystal Enhanced Fluorescence

PubMed Central

Chaudhery, Vikram; George, Sherine; Lu, Meng; Pokhriyal, Anusha; Cunningham, Brian T.

2013-01-01

Photonic crystal (PC) surfaces have been demonstrated as a compelling platform for improving the sensitivity of surface-based fluorescent assays used in disease diagnostics and life science research. PCs can be engineered to support optical resonances at specific wavelengths at which strong electromagnetic fields are utilized to enhance the intensity of surface-bound fluorophore excitation. Meanwhile, the leaky resonant modes of PCs can be used to direct emitted photons within a narrow range of angles for more efficient collection by a fluorescence detection system. The multiplicative effects of enhanced excitation combined with enhanced photon extraction combine to provide improved signal-to-noise ratios for detection of fluorescent emitters, which in turn can be used to reduce the limits of detection of low concentration analytes, such as disease biomarker proteins. Fabrication of PCs using inexpensive manufacturing methods and materials that include replica molding on plastic, nano-imprint lithography on quartz substrates result in devices that are practical for single-use disposable applications. In this review, we will describe the motivation for implementing high-sensitivity fluorescence detection in the context of molecular diagnosis and gene expression analysis though the use of PC surfaces. Recent efforts to improve the design and fabrication of PCs and their associated detection instrumentation are summarized, including the use of PCs coupled with Fabry-Perot cavities and external cavity lasers. PMID:23624689

[Acidity and temperature effect on the fluorescence characteristics of hydraulic oils and lubricants].

PubMed

Deng, Hu; Zhou, Xun; Shang, Li-ping; Zhang, Ze-lin; Wang, Shun-li

2014-12-01

By analyzing HyJet V phosphate ester hydraulic oil environmental impacts (oil, etc.) and confounding factors (pH, temperature, etc.), the feasibility was studied for the fluorescence detection of aircraft hydraulic oil leaks. By using the fluorescence spectrophotometer at various acidities and temperatures, the fluorescence properties of HyJet V phosphate ester hydraulic oil, Jet Oil II lubricant and 2197 lubricant were gained. The experimental results are shown as following: The fluorescence peaks of HyJet V phosphate ester hydraulic oil, Jet Oil II lubricant and 2197 lubricant are at 362, 405 and 456 nm, respectively. The impact of temperature on HyJet V phosphate ester hydraulic oil is less effective; Jet Oil II lubricant and 2197 lubricant fluorescence intensity decreases with increasing temperature. When acidity increases, the fluorescence peak of HyJet V phosphate ester hydraulic oil gradient shifts from 370 to 362 nm, and the fluorescence intensity decreases; the fluorescence peak of Jet Oil II lubricant is always 405 nm, while the fluorescence intensity decreases; the fluorescence peak of 2197 lubricant at 456 nm red shifts to 523 nm, and double fluorescence peaks appeare. The results are shown as following: under the influence of the environment and interference factors, the fluorescence characteristics of HyJet V phosphate ester hydraulic oil remain unchanged, and distinguish from Jet Oil II lubricant and 2197 lubricant. Therefore, the experiments indicate that the detection of HyJet V phosphate ester hydraulic oil leak is feasible by using fluorescence method.

Optimization of mNeonGreen for Homo sapiens increases its fluorescent intensity in mammalian cells.

PubMed

Tanida-Miyake, Emiko; Koike, Masato; Uchiyama, Yasuo; Tanida, Isei

2018-01-01

Green fluorescent protein (GFP) is tremendously useful for investigating many cellular and intracellular events. The monomeric GFP mNeonGreen is about 3- to 5-times brighter than GFP and monomeric enhanced GFP and shows high photostability. The maturation half-time of mNeonGreen is about 3-fold faster than that of monomeric enhanced GFP. However, the cDNA sequence encoding mNeonGreen contains some codons that are rarely used in Homo sapiens. For better expression of mNeonGreen in human cells, we synthesized a human-optimized cDNA encoding mNeonGreen and generated an expression plasmid for humanized mNeonGreen under the control of the cytomegalovirus promoter. The resultant plasmid was introduced into HEK293 cells. The fluorescent intensity of humanized mNeonGreen was about 1.4-fold higher than that of the original mNeonGreen. The humanized mNeonGreen with a mitochondria-targeting signal showed mitochondrial distribution of mNeonGreen. We further generated an expression vector of humanized mNeonGreen with 3xFLAG tags at its carboxyl terminus as these tags are useful for immunological analyses. The 3xFLAG-tagged mNeonGreen was recognized well with an anti-FLAG-M2 antibody. These plasmids for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG are useful tools for biological studies in mammalian cells using mNeonGreen.

Multiple emulsions as effective platforms for controlled anti-cancer drug delivery.

PubMed

Dluska, Ewa; Markowska-Radomska, Agnieszka; Metera, Agata; Tudek, Barbara; Kosicki, Konrad

2017-09-01

Developing pH-responsive multiple emulsion platforms for effective glioblastoma multiforme therapy with reduced toxicity, a drug release study and modeling. Cancer cell line: U87 MG, multiple emulsions with pH-responsive biopolymer and encapsulated doxorubicin (DOX); preparation of multiple emulsions in a Couette-Taylor flow biocontactor, in vitro release study of DOX (fluorescence intensity analysis), in vitro cytotoxicity study (alamarBlue cell viability assay) and numerical simulation of DOX release rates. The multiple emulsions offered a high DOX encapsulation efficiency (97.4 ± 1%) and pH modulated release rates of a drug. Multiple emulsions with a low concentration of DOX (0.02 μM) exhibited broadly advanced cell (U87 MG) cytotoxicity than free DOX solution used at the same concentration. Emulsion platforms could be explored for potential delivery of chemotherapeutics in glioblastoma multiforme therapy.

Fluorescence dynamics of human epidermis (ex vivo) and skin (in vivo)

NASA Astrophysics Data System (ADS)

Salomatina, Elena V.; Pravdin, Alexander B.

2003-10-01

The temporal behavior of autofluorescence of human skin and epidermis under continuous UV-irradiation has been studied. Fluorescence spectra and kinetic curves of fluorescence intensity have been obtained. The fluorescence intensity recovery after dark period also has been examined. The vitiligo skin and epidermis were used for comparing their spectra with reflectance and fluorescence spectra of healthy skin. The epidermal samples were prepared using surface epidermis stripping technique. It has been concluded that fluorophores being undergone the UVA photobleaching are actually present in epidermal layer, and immediate pigment darkening does contribute, no less than a half of magnitude, to the autofluorescence decrease under continuous UVA irradiation.

Confocal fluorescence techniques in industrial application

NASA Astrophysics Data System (ADS)

Eggeling, Christian; Gall, Karsten; Palo, Kaupo; Kask, Peet; Brand, Leif

2003-06-01

The FCS+plus family of evaluation tools for confocal fluorescence spectroscopy, which was developed during recent years, offers a comprehensive view to a series of fluorescence properties. Originating in fluorescence correlation spectroscopy (FCS) and using similar experimental equipment, a system of signal processing methods such as fluorescence intensity distribution analysis (FIDA) was created to analyze in detail the fluctuation behavior of fluorescent particles within a small area of detection. Giving simultaneous access to molecular parameters like concentration, translational and rotational diffusion, molecular brightness, and multicolor coincidence, this portfolio was enhanced by more traditional techniques of fluorescence lifetime as well as time-resolved anisotropy determination. The cornerstones of the FCS+plus methodology will be shortly described. The inhibition of a phosphatase enzyme activity gives a comprehensive industrial application that demonstrates FCS+plus' versatility and its potential for pharmaceutical drug discovery.

Rigidifying fluorescent linkers by metal-organic framework formation for fluorescence blue shift and quantum yield enhancement.

PubMed

Wei, Zhangwen; Gu, Zhi-Yuan; Arvapally, Ravi K; Chen, Ying-Pin; McDougald, Roy N; Ivy, Joshua F; Yakovenko, Andrey A; Feng, Dawei; Omary, Mohammad A; Zhou, Hong-Cai

2014-06-11

We demonstrate that rigidifying the structure of fluorescent linkers by structurally constraining them in metal-organic frameworks (MOFs) to control their conformation effectively tunes the fluorescence energy and enhances the quantum yield. Thus, a new tetraphenylethylene-based zirconium MOF exhibits a deep-blue fluorescent emission at 470 nm with a unity quantum yield (99.9 ± 0.5%) under Ar, representing ca. 3600 cm(-1) blue shift and doubled radiative decay efficiency vs the linker precursor. An anomalous increase in the fluorescence lifetime and relative intensity takes place upon heating the solid MOF from cryogenic to ambient temperatures. The origin of these unusual photoluminescence properties is attributed to twisted linker conformation, intramolecular hindrance, and framework rigidity.

Noninvasive control of the transport function of fluorescent coloured liposomal nanoparticles

NASA Astrophysics Data System (ADS)

Stelmashchuk, O.; Zherebtsov, E.; Zherebtsova, A.; Kuznetsova, E.; Vinokurov, A.; Dunaev, A.; Mamoshin, A.; Snimshchikova, I.; Borsukov, A.; Bykov, A.; Meglinski, I.

2017-06-01

The use of liposomal nanoparticles with an incorporated active substance is an innovative and promising approach to diagnostics and therapy. The application of liposomal nanoparticle-based drugs allows for targeted localized delivery, overcomes the natural barriers within the body effectively, and minimizes possible side effects. Liposomes are able to contain a variety of ingredients with practically no limitations to their chemical composition, chemical properties, or size of constituent molecules. This study evaluated the ability to control the passage of fluorescent dye-filled liposomes through the intestinal mucosal barrier after oral administration. For this purpose, the increase in transcutaneous registered fluorescence from tetrabromofluorescein dye was recorded and analysed. Fluorescence intensity was measured at the proximal end of the tail of an animal model after oral administration of the liposomes. Measurements were taken at the excitation wavelengths of 365 and 450 nm. The fluorescence intensity in the group treated with the fluorescent contrast agent encapsulated in liposomal particles increased 140% of the initial level, but in the group treated with pure contrast agent, the increase in detected fluorescence intensity did not exceed 110%. Mice that received empty liposomes as well as the control group did not demonstrate statistically significant changes in fluorescence intensity. A potential application of our results is an express laser optical method of monitoring the transport of orally administered liposomal particles. The results can be used to help create new optical tools for use in the development of new drugs and in high-throughput screening used during their testing.

Multiphoton fluorescence lifetime imaging of chemotherapy distribution in solid tumors

NASA Astrophysics Data System (ADS)

Carlson, Marjorie; Watson, Adrienne L.; Anderson, Leah; Largaespada, David A.; Provenzano, Paolo P.

2017-11-01

Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.

A PDMS-based cylindrical hybrid lens for enhanced fluorescence detection in microfluidic systems.

PubMed

Lin, Bor-Shyh; Yang, Yu-Ching; Ho, Chong-Yi; Yang, Han-Yu; Wang, Hsiang-Yu

2014-02-13

Microfluidic systems based on fluorescence detection have been developed and applied for many biological and chemical applications. Because of the tiny amount of sample in the system; the induced fluorescence can be weak. Therefore, most microfluidic systems deploy multiple optical components or sophisticated equipment to enhance the efficiency of fluorescence detection. However, these strategies encounter common issues of complex manufacturing processes and high costs. In this study; a miniature, cylindrical and hybrid lens made of polydimethylsiloxane (PDMS) to improve the fluorescence detection in microfluidic systems is proposed. The hybrid lens integrates a laser focusing lens and a fluorescence collecting lens to achieve dual functions and simplify optical setup. Moreover, PDMS has advantages of low-cost and straightforward fabrication compared with conventional optical components. The performance of the proposed lens is first examined with two fluorescent dyes and the results show that the lens provides satisfactory enhancement for fluorescence detection of Rhodamine 6G and Nile Red. The overall increments in collected fluorescence signal and detection sensitivity are more than 220% of those without lens, and the detection limits of Rhodamine 6G and Nile red are lowered to 0.01 μg/mL and 0.05 μg/mL, respectively. The hybrid lens is further applied to the detection of Nile red-labeled Chlorella vulgaris cells and it increases both signal intensity and detection sensitivity by more than 520%. The proposed hybrid lens also dramatically reduces the variation in detected signal caused by the deviation in incident angle of excitation light.

Chlorophyll Fluorescence Analysis of Cyanobacterial Photosynthesis and Acclimation

PubMed Central

Campbell, Douglas; Hurry, Vaughan; Clarke, Adrian K.; Gustafsson, Petter; Öquist, Gunnar

1998-01-01

Cyanobacteria are ecologically important photosynthetic prokaryotes that also serve as popular model organisms for studies of photosynthesis and gene regulation. Both molecular and ecological studies of cyanobacteria benefit from real-time information on photosynthesis and acclimation. Monitoring in vivo chlorophyll fluorescence can provide noninvasive measures of photosynthetic physiology in a wide range of cyanobacteria and cyanolichens and requires only small samples. Cyanobacterial fluorescence patterns are distinct from those of plants, because of key structural and functional properties of cyanobacteria. These include significant fluorescence emission from the light-harvesting phycobiliproteins; large and rapid changes in fluorescence yield (state transitions) which depend on metabolic and environmental conditions; and flexible, overlapping respiratory and photosynthetic electron transport chains. The fluorescence parameters FV/FM, FV′/FM′,qp,qN, NPQ, and φPS II were originally developed to extract information from the fluorescence signals of higher plants. In this review, we consider how the special properties of cyanobacteria can be accommodated and used to extract biologically useful information from cyanobacterial in vivo chlorophyll fluorescence signals. We describe how the pattern of fluorescence yield versus light intensity can be used to predict the acclimated light level for a cyanobacterial population, giving information valuable for both laboratory and field studies of acclimation processes. The size of the change in fluorescence yield during dark-to-light transitions can provide information on respiration and the iron status of the cyanobacteria. Finally, fluorescence parameters can be used to estimate the electron transport rate at the acclimated growth light intensity. PMID:9729605

An artificial tongue fluorescent sensor array for identification and quantitation of various heavy metal ions.

PubMed

Xu, Wang; Ren, Changliang; Teoh, Chai Lean; Peng, Juanjuan; Gadre, Shubhankar Haribhau; Rhee, Hyun-Woo; Lee, Chi-Lik Ken; Chang, Young-Tae

2014-09-02

Herein, a small-molecule fluorescent sensor array for rapid identification of seven heavy metal ions was designed and synthesized, with its sensing mechanism mimicking that of a tongue. The photoinduced electron transfer and intramolecular charge transfer mechanism result in combinatorial interactions between sensor array and heavy metal ions, which lead to diversified fluorescence wavelength shifts and emission intensity changes. Upon principle component analysis (PCA), this result renders clear identification of each heavy metal ion on a 3D spatial dispersion graph. Further exploration provides a concentration-dependent pattern, allowing both qualitative and quantitative measurements of heavy metal ions. On the basis of this information, a "safe-zone" concept was proposed, which provides rapid exclusion of versatile hazardous species from clean water samples based on toxicity characteristic leaching procedure standards. This type of small-molecule fluorescent sensor array could open a new avenue for multiple heavy metal ion detection and simplified water quality analysis.

Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

PubMed Central

Bulina, Maria E; Chudakov, Dmitry M; Mudrik, Nikolay N; Lukyanov, Konstantin A

2002-01-01

Background Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. Conclusions We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed. PMID:11972899

Seeing the electroporative uptake of cell-membrane impermeable fluorescent molecules and nanoparticles

NASA Astrophysics Data System (ADS)

Kim, Kisoo; Kim, Jeong Ah; Lee, Soon-Geul; Lee, Won Gu

2012-07-01

This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities that occurred at cell membranes in both uptake directions toward the electrodes have been sequentially recorded and quantitatively analyzed pixel by pixel. In our experiments, we found that fluorescent molecules, even not labeled to target biomolecules, had their own uptake direction with different intensities. It is also observed that the uptake intensity toward the cell membrane had a maximal value at a certain electric voltage, not at the highest value of voltages applied. The results also imply that the uptake direction of fluorescence-doped nanoparticles can be determined by a net surface charge of uptake materials and sizes in the electroporative environments. In summary, we performed a quantitative screening and direct visualization of uptake directionality for a set of fluorescent molecules and fluorescence-doped nanoparticles using electric-pulsation. Taking a closer look at the uptake direction of exogenous materials will help researchers to understand an unknown uptake phenomenon in which way foreign materials are inclined to move, and furthermore to design functional nanoparticles for electroporative gene delivery.This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities

Multi-spectral endogenous fluorescence imaging for bacterial differentiation

NASA Astrophysics Data System (ADS)

Chernomyrdin, Nikita V.; Babayants, Margarita V.; Korotkov, Oleg V.; Kudrin, Konstantin G.; Rimskaya, Elena N.; Shikunova, Irina A.; Kurlov, Vladimir N.; Cherkasova, Olga P.; Komandin, Gennady A.; Reshetov, Igor V.; Zaytsev, Kirill I.

2017-07-01

In this paper, the multi-spectral endogenous fluorescence imaging was implemented for bacterial differentiation. The fluorescence imaging was performed using a digital camera equipped with a set of visual bandpass filters. Narrowband 365 nm ultraviolet radiation passed through a beam homogenizer was used to excite the sample fluorescence. In order to increase a signal-to-noise ratio and suppress a non-fluorescence background in images, the intensity of the UV excitation was modulated using a mechanical chopper. The principal components were introduced for differentiating the samples of bacteria based on the multi-spectral endogenous fluorescence images.

Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

PubMed

Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

2014-05-01

Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

The NPe6 fluorescence measurements by using a fluorescence sensing system for skin photosensitivity risk assessment after photodynamic therapy

NASA Astrophysics Data System (ADS)

Ohtani, Keishi; Usuda, Jitsu; Ogawa, Emiyu; Maehara, Sachio; Imai, Kentaro; Inoue, Tatsuya; Hagiwara, Masaru; Kakihana, Masatoshi; Kajiwara, Naohiro; Ohira, Tatsuo; Arai, Tsunenori; Ikeda, Norihiko

2018-02-01

Background: Skin photosensitivity is a major side effect of photodynamic therapy (PDT). It is induced by the photosensitizer remaining in the skin. It is usually rapidly metabolized by the liver, but the pharmacokinetic profile varies widely among individuals. This makes it difficult to predict the incidence of skin photosensitivity. Therefore, we conducted a prospective study to investigate whether the NPe6 fluorescence intensity in skin after NPe6-PDT could be measured safely in human patients using a fluorescence sensing system for judging the risk of skin photosensitivity. Methods: The NPe6 fluorescence measurements using a constructed fluorescence sensing system at the inside of the arm were acquired prior to and 5 and 10 minutes after NPe6 administration as well as at the time of PDT (4-5 hours after administration), at discharge (2 or 3 days after PDT), and at 1 or 2 weeks after PDT. Participants were interviewed as to whether they had any complications at 2 weeks after PDT. Results: Nine male patients and one female patient entered this study. All of the measurements of NPe6 fluorescence in the skin could be obtained without any complications. The spectral peak was detected at the time of discharge (2-3 days after administration) in most cases and it decreased at 1 or 2 weeks after PDT. Conclusions: The fluorescence of NPe6 in the skin could be detected feasibly using the fluorescence sensing system in human patients. Measuring fluorescence intensity in the skin might be useful to predict the incidence of skin photosensitivity after PDT.

In-Vivo Fluorescence Spectroscopy Of Normal And Atherosclerotic Arteries

NASA Astrophysics Data System (ADS)

Deckelbaum, Lawrence I.; Sarembock, Ian J.; Stetz, Mark L.; O'Brien, Kenneth M.; Cutruzzola, Francis W.; Gmitro, Arthur F.; Ezekowitz, Michael D.

1988-06-01

Laser-induced fluorescence spectroscopy can discriminate atherosclerotic from normal arteries in-vitro and may thus potentially guide laser angioplasty. To evaluate the feasibility of laser-induced fluorescence spectroscopy in a living blood-filled arterial system we performed fiberoptic laser-induced fluorescence spectroscopy in a rabbit model of focal femoral atherosclerosis. A laser-induced fluorescence spectroscopy score was derived from stepwise linear regression analysis of in-vitro spectra to distinguish normal aorta (score>0) from atherosclerotic femoral artery (score<0). A 400 u silica fiber, coupled to a helium cadmium laser and optical multichannel analyzer, was inserted through a 5F catheter to induce and record in-vivo fluorescence from femoral and aortoiliac arteries. Arterial spectra could be recorded in all animals (n=10: 5 occlusions, 5 stenoses). Blood spectra were of low intensity and were easily distinguished from arterial spectra. The scores (mean ± SEM) for the in-vivo spectra were -0.69 +/- 0.29 for artherosclerotic femoral, and +0.54 ±. 0.15 for normal aorta (p<.01 p=NS compared to in-vitro spectra). In-vitro, a fiber tip to tissue distance <50 u was necessary for adequate arterial LIFS in blood. At larger distances low intensity blood spectra were recorded (1/20 the intensity of tissue spectra). Thus, fiberoptic laser-induced fluorescence spectroscopy can be sucessfully performed in a blood filled artery provided the fiber tip is approximated to the tissue.

Multispectral open-air intraoperative fluorescence imaging.

PubMed

Behrooz, Ali; Waterman, Peter; Vasquez, Kristine O; Meganck, Jeff; Peterson, Jeffrey D; Faqir, Ilias; Kempner, Joshua

2017-08-01

Intraoperative fluorescence imaging informs decisions regarding surgical margins by detecting and localizing signals from fluorescent reporters, labeling targets such as malignant tissues. This guidance reduces the likelihood of undetected malignant tissue remaining after resection, eliminating the need for additional treatment or surgery. The primary challenges in performing open-air intraoperative fluorescence imaging come from the weak intensity of the fluorescence signal in the presence of strong surgical and ambient illumination, and the auto-fluorescence of non-target components, such as tissue, especially in the visible spectral window (400-650 nm). In this work, a multispectral open-air fluorescence imaging system is presented for translational image-guided intraoperative applications, which overcomes these challenges. The system is capable of imaging weak fluorescence signals with nanomolar sensitivity in the presence of surgical illumination. This is done using synchronized fluorescence excitation and image acquisition with real-time background subtraction. Additionally, the system uses a liquid crystal tunable filter for acquisition of multispectral images that are used to spectrally unmix target fluorescence from non-target auto-fluorescence. Results are validated by preclinical studies on murine models and translational canine oncology models.

A label-free fluorescent aptamer sensor based on regulation of malachite green fluorescence

PubMed Central

Xu, Weichen; Lu, Yi

2009-01-01

We report a label-free fluorescent aptamer sensor for adenosine based on the regulation of malachite green (MG) fluorescence, with comparable sensitivity and selectivity to other labeled adenosine aptamer-based sensors. The sensor consists of free MG, an aptamer strand containing an adenosine aptamer next to an MG aptamer, and a bridging strand that partially hybridizes to the aptamer strand. Such a hybridization prevents MG from binding to MG aptamer, resulting in low fluorescence of MG in the absence of adenosine. Addition of adenosine causes the adenosine aptamer to bind adenosine, weakening the hybridization of the aptamer strand with the bridging strand, making it possible for MG to bind to the aptamer strand and exhibits high fluorescence intensity. Since this design is based purely on nucleic acid hybridization, it can be generally applied to other aptamers for the label-free detection of a broad range of analytes. PMID:20017558

Multiple light scattering in metallic ejecta produced under intense shockwave compression.

PubMed

Franzkowiak, J-E; Mercier, P; Prudhomme, G; Berthe, L

2018-04-10

A roughened metallic plate, subjected to intense shock wave compression, gives rise to an expanding ejecta particle cloud. Photonic Doppler velocimetry (PDV), a fiber-based heterodyne velocimeter, is often used to track ejecta velocities in dynamic compression experiments and on nanosecond time scales. Shortly after shock breakout at the metal-vacuum interface, a particular feature observed in many experiments in the velocity spectrograms is what appear to be slow-moving ejecta, below the free-surface velocity. Using Doppler Monte Carlo simulations incorporating the transport of polarization in the ejecta, we show that this feature is likely to be explained by the multiple scattering of light, rather than by possible collisions among particles, slowing down the ejecta. As the cloud expands in a vacuum, the contribution of multiple scattering decreases due to the limited field of view of the pigtailed collimator used to probe the ejecta, showing that the whole geometry of the system must be taken into account in the calculations to interpret and predict PDV measurements.

DNA nanostructure-based fluorescence thermometer with silver nanoclusters

NASA Astrophysics Data System (ADS)

Bu, Congcong; Mu, Lixuan; Cao, Xingxing; Chen, Min; She, Guangwei; Shi, Wensheng

2018-07-01

DNA nanostructure-based fluorescence thermometers were fabricated by linking fluorescent silver nanoclusters (AgNCs) and guanine-rich（G-rich）DNA chains via a thermally sensitive DNA stem-loop at terminals 5‧ and 3‧. Variations of temperature alter the distance between the AgNCs and G-rich DNA chain, affecting the interaction between them. As a result, the intensity of fluorescence emission from the AgNCs at 636 nm can be sensitively modulated. It was found that the intensity of such red emission is more temperature sensitive than the equivalent green emission at 543 nm; sensitivity of ‑3.6%/°C was achieved. Through variation of the melting temperature of the DNA stem-loop, the response temperature range of the thermometers could be readily adjusted. Novel DNA nanostructure-based fluorescence thermometers as described in this work are anticipated to be able to measure the temperature of biological systems at small scales—even a single cell.

Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics.

PubMed

Davis, Caitlin M; Reddish, Michael J; Dyer, R Brian

2017-05-05

Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of <0.2mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50ns to 0.5ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics

NASA Astrophysics Data System (ADS)

Davis, Caitlin M.; Reddish, Michael J.; Dyer, R. Brian

2017-05-01

Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of < 0.2 mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50 ns to 0.5 ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics.

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

Laser induced fluorescence of dental caries

NASA Technical Reports Server (NTRS)

Albin, S.; Byvik, C. E.; Buoncristiani, A. M.

1988-01-01

Significant differences between the optical spectra taken from sound regions of teeth and carious regions have been observed. These differences appear both in absorption and in laser induced fluorescence spectra. Excitation by the 488 nm line of an argon ion laser beam showed a peak in the emission intensity around 553 nm for the sound dental material while the emission peak from the carious region was red-shifted by approximately 40 nm. The relative absorption of carious region was significantly higher at 488 nm; however its fluorescence intensity peak was lower by an order of magnitude compared to the sound tooth. Implications of these results for a safe, reliable and early detection of dental caries are discussed.

Polyester Fabric's Fluorescent Dyeing in Supercritical Carbon Dioxide and its Fluorescence Imaging.

PubMed

Xiong, Xiaoqing; Xu, Yanyan; Zheng, Laijiu; Yan, Jun; Zhao, Hongjuan; Zhang, Juan; Sun, Yanfeng

2017-03-01

As one of the most important coumarin-like dyes, disperse fluorescent Yellow 82 exhibits exceptionally large two-photon effects. Here, it was firstly introduced into the supercritical CO 2 dyeing polyester fabrics in this work. Results of the present work showed that the dyeing parameters such as the dyeing time, pressure and temperature had remarkable influences on the color strength of fabrics. The optimized dyeing condition in supercritical CO 2 dyeing has been proposed that the dyeing time was 60 min; the pressure was 25 MPa and the temperature was 120 °C. As a result, acceptable products were obtained with the wash and rub fastness rating at 5 or 4-5. The polyester fabrics dyed with fluorescent dyes can be satisfied for the requirement of manufacturing warning clothing. Importantly, the confocal microscopy imaging technology was successfully introduced into textile fields to observe the distribution and fluorescence intensity of disperse fluorescent Yellow 82 on polyester fabrics. As far as we know, this is the first report about supercritical CO 2 dyeing polyester fabrics based on disperse fluorescent dyes. It will be very helpful for the further design of new fluorescent functional dyes suitable for supercritical CO 2 dyeing technique.

NIST/ISAC standardization study: variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads.

PubMed

Hoffman, Robert A; Wang, Lili; Bigos, Martin; Nolan, John P

2012-09-01

Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue™), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned

Contribution of glue layer into epidermis sample fluorescence dynamics

NASA Astrophysics Data System (ADS)

Salomatina, Elena V.; Chernova, Svetlana P.; Pravdin, Alexander B.

2000-04-01

In this work, the temporal behavior of autofluorescence of epidermis samples under UV-irradiation has ben studied. The samples were prepared using surface epidermis stripping technique. Fluorescence spectra and kinetic curves of fluorescence intensity have been obtained. It has been concluded that the glue composition used allows the measurement of epidermis fluorescence dynamics with the first 60 min of experiment.

A novel fluorescent retrograde neural tracer: cholera toxin B conjugated carbon dots

NASA Astrophysics Data System (ADS)

Zhou, Nan; Hao, Zeyu; Zhao, Xiaohuan; Maharjan, Suraj; Zhu, Shoujun; Song, Yubin; Yang, Bai; Lu, Laijin

2015-09-01

The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde tracers.The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde

Variations in the endogenous fluorescence of rabbit corneas after mechanical property alterations

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Touchette, Genna; Zhu, Hong; Kochevar, Irene E.; Franco, Walfre

2017-09-01

Keratoconus is an eye disease in which the cornea progressively deforms due to loss of cornea mechanical rigidity, and thus causes deterioration of visual acuity. Techniques to characterize the mechanical characteristics of the cornea are important to better monitor changes and response to treatments. To investigate the feasibility of using the endogenous fluorescence of cornea for monitoring alterations of its mechanical rigidity, linear tensiometry was used to quantitate stiffness and Young's modulus (YM) after treatments that increase cornea stiffness (collagen photocross-linking) or decrease stiffness (enzymatic digestion). The endogenous ultraviolet fluorescence of cornea was also measured before and after these treatments. The fluorescence excitation/emission spectral ranges were 280 to 430/390 to 520 nm, respectively. A correlation analysis was carried out to identify fluorescence excitation/emission pairs whose intensity changes correlated with the stiffness. A positive correlation was found between variations in fluorescence intensity of the 415-/485-nm excitation/emission pair and YM of photocross-linked corneas. After treatment of corneas with pepsin, the YM decreased as the fluorescence intensity at 290-/390-nm wavelengths decreased. For weakening of corneas with collagenase, only qualitative changes in the fluorescence spectrum were observed. Changes in the concentration of native or newly created fluorescent molecular species contain information that may be directly or indirectly related to the mechanical structure of the cornea.

Red Fluorescent Line Emission from Hydrogen Molecules in Diffuse Molecular Clouds

NASA Technical Reports Server (NTRS)

Neufeld, David A.; Spaans, Marco

1996-01-01

We have modeled the fluorescent pumping of electronic and vibrational emissions of molecular hydrogen (H2) within diffuse molecular clouds that are illuminated by ultraviolet continuum radiation. Fluorescent line intensities are predicted for transitions at ultraviolet, infrared, and red visible wavelengths as functions of the gas density, the visual extinction through the cloud, and the intensity of the incident UV continuum radiation. The observed intensity in each fluorescent transition is roughly proportional to the integrated rate of H2 photodissociation along the line of sight. Although the most luminous fluorescent emissions detectable from ground-based observatories lie at near-infrared wavelengths, we argue that the lower sky brightness at visible wavelengths makes the red fluorescent transitions a particularly sensitive probe. Fabry-Perot spectrographs of the type that have been designed to observe very faint diffuse Ha emissions are soon expected to yield sensitivities that will be adequate to detect H2 vibrational emissions from molecular clouds that are exposed to ultraviolet radiation no stronger than the mean radiation field within the Galaxy. Observations of red H2 fluorescent emission together with cospatial 21 cm H I observations could serve as a valuable probe of the gas density in diffuse molecular clouds.

Construction of a 'turn-on' fluorescent probe system for His-tagged proteins.

PubMed

Murata, Atsushi; Arai, Satoshi; Yoon, Su-In; Takabayashi, Masao; Ozaki, Miwako; Takeoka, Shinji

2010-12-01

Hexahistidine ((His)(6)) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)(6), and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni(2+), can bind (His)(6). The system is turned off when Dabcyl-(His)(6) is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)(6)-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)(6)-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His)(6)-protein. Copyright © 2010 Elsevier Ltd. All rights reserved.

A fluorescent molecular rotor probes the kinetic process of degranulation of mast cells.

PubMed

Furuno, T; Isoda, R; Inagaki, K; Iwaki, T; Noji, M; Nakanishi, M

1992-08-01

A confocal fluorescence microscope was used to study the exocytotic secretory processes of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its quantum yield with decreasing intramolecular rotation. Here, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramatically in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D (10 microM). However, fluo-3 fluorescence intensities still increased after addition of compound 48/80.

Detecting crop population growth using chlorophyll fluorescence imaging.

PubMed

Wang, Heng; Qian, Xiangjie; Zhang, Lan; Xu, Sailong; Li, Haifeng; Xia, Xiaojian; Dai, Liankui; Xu, Liang; Yu, Jingquan; Liu, Xu

2017-12-10

For both field and greenhouse crops, it is challenging to evaluate their growth information on a large area over a long time. In this work, we developed a chlorophyll fluorescence imaging-based system for crop population growth information detection. Modular design was used to make the system provide high-intensity uniform illumination. This system can perform modulated chlorophyll fluorescence induction kinetics measurement and chlorophyll fluorescence parameter imaging over a large area of up to 45  cm×34  cm. The system can provide different lighting intensity by modulating the duty cycle of its control signal. Results of continuous monitoring of cucumbers in nitrogen deficiency show the system can reduce the judge error of crop physiological status and improve monitoring efficiency. Meanwhile, the system is promising in high throughput application scenarios.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

PubMed Central

Pathak, Rupak; Koturbash, Igor; Hauer-Jensen, Martin

2017-01-01

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics. PMID:28117817

Critical analysis of commonly used fluorescence metrics to characterize dissolved organic matter.

PubMed

Korak, Julie A; Dotson, Aaron D; Summers, R Scott; Rosario-Ortiz, Fernando L

2014-02-01

The use of fluorescence spectroscopy for the analysis and characterization of dissolved organic matter (DOM) has gained widespread interest over the past decade, in part because of its ease of use and ability to provide bulk DOM chemical characteristics. However, the lack of standard approaches for analysis and data evaluation has complicated its use. This study utilized comparative statistics to systematically evaluate commonly used fluorescence metrics for DOM characterization to provide insight into the implications for data analysis and interpretation such as peak picking methods, carbon-normalized metrics and the fluorescence index (FI). The uncertainty associated with peak picking methods was evaluated, including the reporting of peak intensity and peak position. The linear relationship between fluorescence intensity and dissolved organic carbon (DOC) concentration was found to deviate from linearity at environmentally relevant concentrations and simultaneously across all peak regions. Comparative analysis suggests that the loss of linearity is composition specific and likely due to non-ideal intermolecular interactions of the DOM rather than the inner filter effects. For some DOM sources, Peak A deviated from linearity at optical densities a factor of 2 higher than that of Peak C. For carbon-normalized fluorescence intensities, the error associated with DOC measurements significantly decreases the ability to distinguish compositional differences. An in-depth analysis of FI determined that the metric is mostly driven by peak emission wavelength and less by emission spectra slope. This study also demonstrates that fluorescence intensity follows property balance principles, but the fluorescence index does not. Copyright © 2013 Elsevier Ltd. All rights reserved.

Temperature dependence of the ratio of intensities of up-conversion fluorescence bands of YVO4 and YGdVO4 crystals and lead fluoride nano glass ceramics activated with erbium ions

NASA Astrophysics Data System (ADS)

Varaksa, Yu. A.; Sinitsyn, G. V.; Khodasevich, M. A.; Aseev, V. A.; Kolobkova, E. V.; Yasyukevich, A. S.

2015-01-01

Up-conversion fluorescence spectra of YVO4 and YGdVO4 crystals and lead fluoride nano glass ceramics coactivated with erbium and ytterbium ions have been studied in the wavelength range of 520-560 nm under 967-nm pumping. The ratio of intensities of fluorescence bands in the ranges of 520-530 and 540-550 nm has been measured in the temperature range of from room temperature to 150°C. It is shown that the considered materials can be used for preparing a sensing element of optical fluorescent temperature sensors; the sensitivity of measuring the temperature of nano glass-ceramics can be close to that of crystal samples.

Assessment of tissue ischemia of nail fold precapillary zones using a fluorescence capillaroscopy

NASA Astrophysics Data System (ADS)

Dremin, Viktor V.; Margaryants, Nikita B.; Volkov, Mikhail V.; Zhukova, Ekaterina V.; Zherebtsov, Evgeny A.; Dunaev, Andrey V.; Rafailov, Edik U.

2017-07-01

An optical instrument for nailfold fluorescence capillaroscopy and image registration has been developed. With this instrument, an effect of increasing fluorescence intensity in the spectral range of NADH fluorescence during ischemia was detected.

Optical spectroscopy of the bladder washout fluid to optimize fluorescence cystoscopy with Hexvix®.

PubMed

Martoccia, Carla; Zellweger, Matthieu; Lovisa, Blaise; Jichlinski, Patrice; van den Bergh, Hubert; Wagnières, Georges

2014-09-01

Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate thebladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degradesfluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studiedtheir fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C).A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320∕420 nm, FWHM =50∕100 nm) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455∕525 nm, FWHM = 80∕50 nm). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine’s main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370–430 nm to 395–415 nm would reduce the BWF background by a factor 2.

Fluorescence lifetime as a new parameter in analytical cytology measurements

NASA Astrophysics Data System (ADS)

Steinkamp, John A.; Deka, Chiranjit; Lehnert, Bruce E.; Crissman, Harry A.

1996-05-01

A phase-sensitive flow cytometer has been developed to quantify fluorescence decay lifetimes on fluorochrome-labeled cells/particles. This instrument combines flow cytometry (FCM) and frequency-domain fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved lifetime measurements, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by conventional and phase-sensitive signal detection electronics and displayed as frequency distribution histograms. In this study we describe results of fluorescence intensity and lifetime measurements on fluorescently labeled particles, cells, and chromosomes. Examples of measurements on intrinsic cellular autofluorescence, cells labeled with immunofluorescence markers for cell- surface antigens, mitochondria stains, and on cellular DNA and protein binding fluorochromes will be presented to illustrate unique differences in measured lifetimes and changes caused by fluorescence quenching. This innovative technology will be used to probe fluorochrome/molecular interactions in the microenvironment of cells/chromosomes as a new parameter and thus expand the researchers' understanding of biochemical processes and structural features at the cellular and molecular level.

Adjustable repetition-rate multiplication of optical pulses using fractional temporal Talbot effect with preceded binary intensity modulation

NASA Astrophysics Data System (ADS)

Xie, Qijie; Zheng, Bofang; Shu, Chester

2017-05-01

We demonstrate a simple approach for adjustable multiplication of optical pulses in a fiber using the temporal Talbot effect. Binary electrical patterns are used to control the multiplication factor in our approach. The input 10 GHz picosecond pulses are pedestal-free and are shaped directly from a CW laser. The pulses are then intensity modulated by different sets of binary patterns prior to entering a fiber of fixed dispersion. Tunable repetition-rate multiplication by different factors of 2, 4, and 8 have been achieved and up to 80 GHz pulse train has been experimentally generated. We also evaluate numerically the influence of the extinction ratio of the intensity modulator on the performance of the multiplied pulse train. In addition, the impact of the modulator bias on the uniformity of the output pulses has also been analyzed through simulation and experiment and a good agreement is reached. Last, we perform numerical simulation on the RF spectral characteristics of the output pulses. The insensitivity of the signal-to-subharmonic noise ratio (SSNR) to the laser linewidth shows that our multiplication scheme is highly tolerant to the incoherence of the input optical pulses.

Laser-Induced Fluorescence (LIF) from plant foliage

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Williams, D. L.

1986-01-01

The fluorescence spectra and fluorescence induction kinetics of green plants excited at 337 nm by a laser were studied. They correlate with plant type, as well as with changes in the physiology of the plant as the result of stress. The plant types studied include herbaceous dicots, monocots, hardwoods, conifers, and algae. These plant types could be identified on the basis of differences in either the number of fluorescent bands or the relative intensity of the bands. Differences in fluorescent spectra which could be related to vigor status are observed in conifers located in an area of high atmospheric deposition. Changes in the fluorescence spectra and induction kinetics are also seen in plants grown under conditions of nutrient deficiency and drought stress.

Laser-Induced Fluorescence (LIF) from plant foliage

NASA Technical Reports Server (NTRS)

Chappelle, Emmett W.; Williams, Darrel L.

1987-01-01

The fluorescence spectra and fluorescence induction kinetics of green plants excited at 337 nm by a laser were studied. They correlate with plant type, as well as with changes in the physiology of the plant as the result of stress. The plant types studied include herbaceous dicots, monocots, hardwoods, conifers, and algae. These plant types could be identified on the basis of differences in either the number of fluorescent bands or the relative intensity of the bands. Differences in fluorescent spectra which could be related to vigor status are observed in conifers located in an area of high atmospheric deposition. Changes in the fluorescence spectra and induction kinetics are also seen in plants grown under conditions of nutrient deficiency and drought stress.

Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

USGS Publications Warehouse

Hackley, Paul C.; Kus, Jolanta

2015-01-01

We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

Computer Modeling of the Structure and Spectra of Fluorescent Proteins

PubMed Central

Grigorenko, B.L.; Savitsky, A.P.

2009-01-01

Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers. PMID:22649601

Infection-Mediated Vasoactive Peptides Modulate Cochlear Uptake of Fluorescent Gentamicin

PubMed Central

Koo, Ja-Won; Wang, Qi; Steyger, Peter S.

2011-01-01

Inflammatory mediators released during bacterial infection include vasoactive peptides such as histamine and serotonin, and their serum levels are frequently elevated. These peptides also modulate the vascular permeability of endothelial cells lining the blood-brain and blood-labyrinth barriers (BLB). These peptides may also modulate the permeability of the BLB to ototoxic aminoglycoside antibiotics prescribed to resolve bacterial sepsis. To test this hypothesis, we compared the effect of histamine and serotonin on the cochlear distribution of fluorescently conjugated gentamicin (GTTR) in control animals at 0.5, 1 and 3 h after injection of GTTR. The intensity of GTTR fluorescence was attenuated at 1 h in the histamine group compared to control mice, and more intense 3 h after injection (p < 0.05). In the serotonin group, the intensity of GTTR fluorescence was attenuated at 0.5 and 1 h (p < 0.05) and was increased at 3 h compared to control animals, where GTTR intensities peaked at 1 h and then plateaued or was slightly decreased at 3 h. This biphasic pattern of modulation was statistically significant in the apical turn of the cochlea. No difference in the intensity of GTTR fluorescence was observed in kidney proximal tubules. Systemic increases in serum levels of vasoactive peptides can modulate cochlear uptake of gentamicin, likely via permeability changes in the BLB. Conditions that influence serum levels of vasoactive peptides may potentiate aminoglycoside ototoxicity. PMID:21196726

Control of excitation in the fluorescence microscope.

PubMed

Lea, D J; Ward, D J

1979-01-01

In fluorescence microscopy image brightness and contrast and the rate of fading depend upon the intensity of illumination of the specimen. An iris diaphragm or neutral density filters may be used to reduce fluorescence excitation. Also the excitation bandwidth may be varied by using a broad band exciter filter with a set of interchangeable yellow glass filters at the lamphouse.

Apparatus for eliminating background interference in fluorescence measurements

DOEpatents

Martin, J.C.; Jett, J.H.

1984-01-06

The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser which excites a different stained component of the same biological particle.

Apparatus for eliminating background interference in fluorescence measurements

DOEpatents

Martin, John C.; Jett, James H.

1986-01-01

The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser, which excites a different stained component of the same biological particle.

Apparatus for eliminating background interference in fluorescence measurements

DOEpatents

Martin, J.C.; Jett, J.H.

1986-03-04

The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser, which excites a different stained component of the same biological particle. 8 figs.

Dilution of protein-surfactant complexes: a fluorescence study.

PubMed

Azadi, Glareh; Chauhan, Anuj; Tripathi, Anubhav

2013-09-01

Dilution of protein-surfactant complexes is an integrated step in microfluidic protein sizing, where the contribution of free micelles to the overall fluorescence is reduced by dilution. This process can be further improved by establishing an optimum surfactant concentration and quantifying the amount of protein based on the fluorescence intensity. To this end, we study the interaction of proteins with anionic sodium dodecyl sulfate (SDS) and cationic hexadecyl trimethyl ammonium bromide (CTAB) using a hydrophobic fluorescent dye (sypro orange). We analyze these interactions fluourometrically with bovine serum albumin, carbonic anhydrase, and beta-galactosidase as model proteins. The fluorescent signature of protein-surfactant complexes at various dilution points shows three distinct regions, surfactant dominant, breakdown, and protein dominant region. Based on the dilution behavior of protein-surfactant complexes, we propose a fluorescence model to explain the contribution of free and bound micelles to the overall fluorescence. Our results show that protein peak is observed at 3 mM SDS as the optimum dilution concentration. Furthermore, we study the effect of protein concentration on fluorescence intensity. In a single protein model with a constant dye quantum yield, the peak height increases with protein concentration. Finally, addition of CTAB to the protein-SDS complex at mole fractions above 0.1 shifts the protein peak from 3 mM to 4 mM SDS. The knowledge of protein-surfactant interactions obtained from these studies provides significant insights for novel detection and quantification techniques in microfluidics. © 2013 The Protein Society.

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes.

PubMed

Model, Michael A; Blank, James L

2006-10-01

To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.

Fluorometric aptasensing of the neonicotinoid insecticide acetamiprid by using multiple complementary strands and gold nanoparticles.

PubMed

Bahreyni, Amirhossein; Yazdian-Robati, Rezvan; Ramezani, Mohammad; Abnous, Khalil; Taghdisi, Seyed Mohammad

2018-04-29

A fluorometric aptamer-based assay was developed for ultrasensitive and selective determination of the neonicotinoid insecticide acetamiprid. The method is based on the use of an aptamer against acetamiprid, multiple complementary strands (CSs), and gold nanoparticles (AuNPs). It is found that by using different CSs, the sensitivity and selectivity of the method is enhanced. On addition of acetamiprid to the aptamer, they will bind to each other and CS1-fluorescein (FAM)-labeled CS2 (as a dsDNA) will be formed. The FAM-labeled dsDNA does not bind to the AuNPs (as a strong quencher) and remains free in the environment, resulting in a strong fluorescence intensity. Without the introduction of acetamiprid, FAM-labeled CS2 binds to AuNPs directly and indirectly through hybridization with CS3 immobilized on the surface of the AuNPs. So, the fluorescence intensity of FAM-labeled CS2 is significantly quenched by AuNPs. The method can detect acetamiprid in the 5 to 50 nM concentration range with a 2.8 nM detection limit. The assay was applied to the determination of acetamiprid in spiked tap water where is gave recoveries that ranged between 95.4% and 94.4%. Graphical abstract (a) In the presence of acetamiprid, aptamer interacts with acetamiprid. The formation of aptamer/acetamiprid causes pairing of complementary strand 1 with FAM-labeled complementary strand, leading to a strong fluorescence intensity. (b) In the absence of acetamiprid, aptamer is hybridized with complementary strand 1. Thus, a very weak fluorescence signal is detected.

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

PubMed

Petersen, Timothy W; Ibrahim, Sherrif F; Diercks, Alan H; van den Engh, Ger

2004-08-01

Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.

A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy.

PubMed

Tiwari, Dhermendra K; Arai, Yoshiyuki; Yamanaka, Masahito; Matsuda, Tomoki; Agetsuma, Masakazu; Nakano, Masahiro; Fujita, Katsumasa; Nagai, Takeharu

2015-06-01

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa

PubMed Central

Vass, H.; Reischl, B.; Allen, R. J.; Friedrich, O.

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules Δdv¯ is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence. PMID:27764134

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

PubMed

Schneidereit, D; Vass, H; Reischl, B; Allen, R J; Friedrich, O

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

Multiplex and high-throughput DNA detection using surface plasmon mediated fluorescence

NASA Astrophysics Data System (ADS)

Mei, Zhong

The overall objective of this research project was to develop a user-friendly and sensitive biosensor for nucleic acid aptamers with multiplexing and high-throughput capability. The sensing was based on the fluorescence signals emitted by the fluorophores coupling with plamonic nanoparticle (gold nanorod) deposited on a patterned substrate. Gold nanorods (GNRs) were synthesized using a binary mixture of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NaOL) in seed mediated growth method. Polytetrafluoroethylene (PTFE) printed glass slides were selectively coated with a gold thin-film to define hydrophilic areas for GNR deposition. Due to the wettablity contrast, GNR solution dropped on the slide was induced to assemble exclusively in the hydrophilic spots. By controlling temperature and humidity of the evaporation process, vertically-standing GNR arrays were achieved on the pattered slide. Fluorescence was conjugated to GNR surface via DNA double strand with tunable length. Theoretical simulation predicted a flat layer ( 30 nm thick) of uniform "hot spots" presented on the GNR tips, which could modify the nearby fluorescence. Experimentally, the vertical GNR arrays yielded metallic enhanced fluorescence (MEF) effect, which was dependent on the spectrum overlap and GNR-fluorophore distance. Specifically, the maximum enhancement of Quasar 670 and Alexa 750 was observed when it was coupled with GNR664 (plasmonic wavelength 664 nm) and GNR778 respectively at a distance of 16 nm, while the carboxyfluorescein (FAM) was at maximal intensity when attached to gold nanosphere520. This offers an opportunity for multiplexed DNA sensing. Based on this, we developed a novel GNR mediated fluorescence biosensor for DNA detection. Fluorescence labeled haipin-DNA probes were introduced to designated spots of GNR array with the matching LSPR wavelengths on the substrate. The fluorescence was quenched originally because of Forster resonance energy transfer (FRET) effect

Hybrid phosphorescence and fluorescence native spectroscopy for breast cancer detection.

PubMed

Alimova, Alexandra; Katz, A; Sriramoju, Vidyasagar; Budansky, Yuri; Bykov, Alexei A; Zeylikovich, Roman; Alfano, R R

2007-01-01

Fluorescence and phosphorescence measurements are performed on normal and malignant ex vivo human breast tissues using UV LED and xenon lamp excitation. Tryptophan (trp) phosphorescence intensity is higher in both normal glandular and adipose tissue when compared to malignant tissue. An algorithm based on the ratio of trp fluorescence intensity at 345 nm to phosphorescence intensity at 500 nm is successfully used to separate normal from malignant tissue types. Normal specimens consistently exhibited a low I(345)I(500) ratio (<10), while for malignant specimens, the I(345)I(500) ratio is consistently high (>15). The ratio analysis correlates well with histopathology. Intensity ratio maps with a spatial resolution of 0.5 mm are generated in which local regions of malignancy could be identified.

Quantification of Coffea arabica and Coffea canephora var. robusta concentration in blends by means of synchronous fluorescence and UV-Vis spectroscopies.

PubMed

Dankowska, A; Domagała, A; Kowalewski, W

2017-09-01

The potential of fluorescence, UV-Vis spectroscopies as well as the low- and mid-level data fusion of both spectroscopies for the quantification of concentrations of roasted Coffea arabica and Coffea canephora var. robusta in coffee blends was investigated. Principal component analysis was used to reduce data multidimensionality. To calculate the level of undeclared addition, multiple linear regression (PCA-MLR) models were used with lowest root mean square error of calibration (RMSEC) of 3.6% and root mean square error of cross-validation (RMSECV) of 7.9%. LDA analysis was applied to fluorescence intensities and UV spectra of Coffea arabica, canephora samples, and their mixtures in order to examine classification ability. The best performance of PCA-LDA analysis was observed for data fusion of UV and fluorescence intensity measurements at wavelength interval of 60nm. LDA showed that data fusion can achieve over 96% of correct classifications (sensitivity) in the test set and 100% of correct classifications in the training set, with low-level data fusion. The corresponding results for individual spectroscopies ranged from 90% (UV-Vis spectroscopy) to 77% (synchronous fluorescence) in the test set, and from 93% to 97% in the training set. The results demonstrate that fluorescence, UV, and visible spectroscopies complement each other, giving a complementary effect for the quantification of roasted Coffea arabica and Coffea canephora var. robusta concentration in blends. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting.

PubMed

Lamb, D C; Müller, B K; Bräuchle, C

2005-10-01

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.

The Origin of Fluorescence from Graphene Oxide

PubMed Central

Shang, Jingzhi; Ma, Lin; Li, Jiewei; Ai, Wei; Yu, Ting; Gurzadyan, Gagik G.

2012-01-01

Time-resolved fluorescence measurements of graphene oxide in water show multiexponential decay kinetics ranging from 1 ps to 2 ns. Electron-hole recombination from the bottom of the conduction band and nearby localized states to wide-range valance band is suggested as origin of the fluorescence. Excitation wavelength dependence of the fluorescence was caused by relative intensity changes of few emission species. By introducing the molecular orbital concept, the dominant fluorescence was found to originate from the electronic transitions among/between the non-oxidized carbon regions and the boundary of oxidized carbon atom regions, where all three kinds of functionalized groups C-O, C = O and O = C-OH were participating. In the visible spectral range, the ultrafast fluorescence of graphene oxide was observed for the first time. PMID:23145316

Double-excitation fluorescence spectral imaging: eliminating tissue auto-fluorescence from in vivo PPIX measurements

NASA Astrophysics Data System (ADS)

Torosean, Sason; Flynn, Brendan; Samkoe, Kimberley S.; Davis, Scott C.; Gunn, Jason; Axelsson, Johan; Pogue, Brian W.

2012-02-01

An ultrasound coupled handheld-probe-based optical fluorescence molecular tomography (FMT) system has been in development for the purpose of quantifying the production of Protoporphyrin IX (PPIX) in aminolevulinic acid treated (ALA), Basal Cell Carcinoma (BCC) in vivo. The design couples fiber-based spectral sampling of PPIX fluorescence emission with a high frequency ultrasound imaging system, allowing regionally localized fluorescence intensities to be quantified [1]. The optical data are obtained by sequential excitation of the tissue with a 633nm laser, at four source locations and five parallel detections at each of the five interspersed detection locations. This method of acquisition permits fluorescence detection for both superficial and deep locations in ultrasound field. The optical boundary data, tissue layers segmented from ultrasound image and diffusion theory are used to estimate the fluorescence in tissue layers. To improve the recovery of the fluorescence signal of PPIX, eliminating tissue autofluorescence is of great importance. Here the approach was to utilize measurements which straddled the steep Qband excitation peak of PPIX, via the integration of an additional laser source, exciting at 637 nm; a wavelength with a 2 fold lower PPIX excitation value than 633nm.The auto-fluorescence spectrum acquired from the 637 nm laser is then used to spectrally decouple the fluorescence data and produce an accurate fluorescence emission signal, because the two wavelengths have very similar auto-fluorescence but substantially different PPIX excitation levels. The accuracy of this method, using a single source detector pair setup, is verified through animal tumor model experiments, and the result is compared to different methods of fluorescence signal recovery.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth

2005-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, 51%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear hits. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Minamitani, Elizabeth Forsythe; Pusey, Marc L.

2004-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of a macromolecules purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals will show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "bits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth; Achari, Amiruddha

2005-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1 %, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth

2004-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically can not reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low

Connecting active to passive fluorescence with photosynthesis: a method for evaluating remote sensing measurements of Chl fluorescence.

PubMed

Magney, Troy S; Frankenberg, Christian; Fisher, Joshua B; Sun, Ying; North, Gretchen B; Davis, Thomas S; Kornfeld, Ari; Siebke, Katharina

2017-09-01

Recent advances in the retrieval of Chl fluorescence from space using passive methods (solar-induced Chl fluorescence, SIF) promise improved mapping of plant photosynthesis globally. However, unresolved issues related to the spatial, spectral, and temporal dynamics of vegetation fluorescence complicate our ability to interpret SIF measurements. We developed an instrument to measure leaf-level gas exchange simultaneously with pulse-amplitude modulation (PAM) and spectrally resolved fluorescence over the same field of view - allowing us to investigate the relationships between active and passive fluorescence with photosynthesis. Strongly correlated, slope-dependent relationships were observed between measured spectra across all wavelengths (F λ , 670-850 nm) and PAM fluorescence parameters under a range of actinic light intensities (steady-state fluorescence yields, F t ) and saturation pulses (maximal fluorescence yields, F m ). Our results suggest that this method can accurately reproduce the full Chl emission spectra - capturing the spectral dynamics associated with changes in the yields of fluorescence, photochemical (ΦPSII), and nonphotochemical quenching (NPQ). We discuss how this method may establish a link between photosynthetic capacity and the mechanistic drivers of wavelength-specific fluorescence emission during changes in environmental conditions (light, temperature, humidity). Our emphasis is on future research directions linking spectral fluorescence to photosynthesis, ΦPSII, and NPQ. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Fluorescence lifetime spectroscopy in multiple-scattering environments: an application to biotechnology

NASA Astrophysics Data System (ADS)

Cerussi, Albert E.; Gratton, Enrico; Fantini, Sergio

1999-07-01

Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in the milk, which is an application vital to the tremendous economic burden of bovine subclinical mastitis detection. Furthermore, we may in principle use the spirit of this technique as a quantitative monitor of the binding of fluorescent drugs inside tissues. This is a first step towards lifetime spectroscopy in tissues.

Tomato seeds maturity detection system based on chlorophyll fluorescence

NASA Astrophysics Data System (ADS)

Li, Cuiling; Wang, Xiu; Meng, Zhijun

2016-10-01

Chlorophyll fluorescence intensity can be used as seed maturity and quality evaluation indicator. Chlorophyll fluorescence intensity of seed coats is tested to judge the level of chlorophyll content in seeds, and further to judge the maturity and quality of seeds. This research developed a detection system of tomato seeds maturity based on chlorophyll fluorescence spectrum technology, the system included an excitation light source unit, a fluorescent signal acquisition unit and a data processing unit. The excitation light source unit consisted of two high power LEDs, two radiators and two constant current power supplies, and it was designed to excite chlorophyll fluorescence of tomato seeds. The fluorescent signal acquisition unit was made up of a fluorescence spectrometer, an optical fiber, an optical fiber scaffolds and a narrowband filter. The data processing unit mainly included a computer. Tomato fruits of green ripe stage, discoloration stage, firm ripe stage and full ripe stage were harvested, and their seeds were collected directly. In this research, the developed tomato seeds maturity testing system was used to collect fluorescence spectrums of tomato seeds of different maturities. Principal component analysis (PCA) method was utilized to reduce the dimension of spectral data and extract principal components, and PCA was combined with linear discriminant analysis (LDA) to establish discriminant model of tomato seeds maturity, the discriminant accuracy was greater than 90%. Research results show that using chlorophyll fluorescence spectrum technology is feasible for seeds maturity detection, and the developed tomato seeds maturity testing system has high detection accuracy.

The Effect of Intense Laser Radiation on Atomic Collisions

NASA Astrophysics Data System (ADS)

Young, Stephen Michael Radley

1991-02-01

Available from UMI in association with The British Library. Requires signed TDF. We have carried out theoretical and experimental studies into the effect of intense laser radiation on atomic collisions. The first experiment used neon. Excitation by electron impact in a gas discharge demanded a pressure of at least 0.075 Torr. Measurement of the intensity of 3^1S_0to 3^1P_1 fluorescence has been made for the case where high intensity ASE wings in the laser profile and background laser scatter are unimportant, with the laser tuned to resonance. The field intensity required to produce strong field fluorescence (exemplified by the Mollow triplet) was found to give rise to complications capable of screening the effects sought. Our theoretical model has suggested that at finite detunings, line-centre fluorescence will dominate Rayleigh scatter and omega_3 fluorescence. Our measurements provide information on the saturation of neon fluorescence but not of the variation of the intense field collision rate. Absorption of weak field 253.7 nm laser photons by ground state mercury atoms yielded a high 6 ^3P_1 population at a lower pressure of 0.02 Torr. The Mollow triplet has been observed in the self-broadened mercury system. Dressing of the upper transition (6^3P_1rightarrow 7^3S_1) by an intense laser close to 435.8 nm yielded the strong field signal. Polarisation studies were made possible by the 3-level mercury system (radiation trapping in a 2-level system would depolarise fluorescence) perturbed by argon. The studies yielded results that were explainable in terms of the selective population of Stark shifted dressed states by a detuned, weak probe field. Use has been made of the electric-dipole radiation selection rule m_{J}=0 rightarrow m_{J^' } = 0 unless J=J^' to devise a 'Stark shift collision switch'. The competition between collision and radiation induced transitions within the mercury atom has then been studied. The resonant, strong lambda 435.8 nm field was used

[Fluorescence characterization of dissolved organic matter in the East China Sea after diatom red tide dispersion].

PubMed

Zhuo, Peng-ji; Zhao, Wei-hong

2009-05-01

Fluorescence excitation-emission spectroscopy (EEMS) was employed to analyze the 3-dimensional fluorescence of dissolved organic matter in the East China Sea after diatom red tide dispersion. The relationships between fluorescence peak intensity, and salinity and chlorophyll-a were discussed. The centers of protein-like fluorescence peaks dispersed at Exmax/Exmax = 270-280/290-315 nm (Peak B), 220-230/290-305 nm (Peak D), 230-240/335-350 nm (Peak S) and 280/320 nm (Peak T). Two humic-like peaks appeared at 255-270/435-480 nm (Peak A)and 330-350/420-480 nm (Peak C). High tyrosine-like intensity was observed in diatom red tide dispersion area, and tryptophan-like fluorescence was also found which was lower. High FIB/FIS showed that diatom red tide produced much tyrosine-like matter during dispersion. Peaks S, A and C had positive correlation with one another, and their distributions were similar, which decreased with distance increasing away from the shore. Good negative correlations between peaks S, A and C and salinity suggested that Jiangsu-Zhejiang coastal water was the same source of them. Correlations between fluorescence peak intensity and chlorophyll-a were not remarkable enough to clear the relationship between fluorescence and living algal matter. It was supposed that the living algal matter contributed little to the fluorescence intensity of algal dispersion seawater.

[Laser Induced Fluorescence Spectroscopic Analysis of Aromatics from One Ring to Four Rings].

PubMed

Zhang, Peng; Liu, Hai-feng; Yue, Zong-yu; Chen, Bei-ling; Yao, Ming-fa

2015-06-01

In order to distinguish small aromatics preferably, a Nd : YAG Laser was used to supply an excitation laser, which was adjusted to 0.085 J x cm(-2) at 266 nm. Benzene, toluene, naphthalene, phenanthrene, anthracene, pyrene and chrysene were used as the representative of different rings aromatics. The fluorescence emission spectra were researched for each aromatic hydrocarbon and mixtures by Laser induced fluorescence (LIF). Results showed that the rings number determined the fluorescence emission spectra, and the structure with same rings number did not affect the emission fluorescence spectrum ranges. This was due to the fact that the absorption efficiency difference at 266 nm resulted in that the fluorescence intensities of each aromatic hydrocarbon with same rings number were different and the fluorescence intensities difference were more apparently with aromatic ring number increasing. When the absorption efficiency was similar at 266 nm and the concentrations of each aromatic hydrocarbon were same, the fluorescence intensities were increased with aromatic ring number increasing. With aromatic ring number increasing, the fluorescence spectrum and emission peak wavelength were all red-shifted from ultraviolet to visible and the fluorescence spectrum range was also wider as the absorption efficiency was similar. The fluorescence emission spectra from one to four rings could be discriminated in the following wavelengths, 275 to 320 nm, 320 to 375 nm, 375 to 425 nm, 425 to 556 nm, respectively. It can be used for distinguish the type of the polycyclic aromatic hydrocarbons (PAHs) as it exists in single type. As PAHs are usually exist in a variety of different rings number at the same time, the results for each aromatic hydrocarbon may not apply to the aromatic hydrocarbon mixtures. For the aromatic hydrocarbon mixtures, results showed that the one- or two-ring PAHs in mixtures could not be detected by fluorescence as three- or four-ring PAHs existed in mixture

Non-destructive mobile monitoring of microbial contaminations on meat surfaces using porphyrin fluorescence intensities.

PubMed

Durek, J; Fröhling, A; Bolling, J; Thomasius, R; Durek, P; Schlüter, O K

2016-05-01

A non-destructive mobile system for meat quality monitoring was developed and investigated for the possible application along the whole production chain of fresh meat. Pork and lamb meat was stored at 5 °C for up to 20 days post mortem and measured with a fluorescence spectrometer. Additionally, the bacterial influence on the fluorescence signals was evaluated by different experimental procedures. Fluorescence of NADH and different porphyrins could be correlated to the growth of diverse bacteria and hence used for contamination monitoring. The increase of porphyrin fluorescence started after 9 days p.m. for pork and after 2 days p.m. for lamb meat. Based on the results, a mobile fluorescence system was built and compared with the laboratory system. The corrected function of the meat slices showed a root mean square error of 1156.97 r.u. and a mean absolute percentage error of 12.59%; for lamb the values were 470.81 r.u. and 15.55%, respectively. A mobile and non-invasive measurement system would improve the microbial security of fresh meat. Copyright © 2016 Elsevier Ltd. All rights reserved.

Al-based metal-organic gels for selective fluorescence recognition of hydroxyl nitro aromatic compounds

NASA Astrophysics Data System (ADS)

Guo, Mao Xia; Yang, Liu; Jiang, Zhong Wei; Peng, Zhe Wei; Li, Yuan Fang

2017-12-01

The novel class of luminescent Al3 +-based metal-organic gels (Al-MOGs) have been developed by mix 4-[2,2‧:6‧,2″-terpyridine]-4‧-ylbenzoic acid (Hcptpy) with Al3 + under mild condition. The as-prepared Al-MOGs have not only multiple stimuli-responsive properties, but selective recognition of hydroxyl nitro aromatic compounds, which can quench the fluorescence of the Al-MOGs, while other nitro aromatic analogues without hydroxyl substitutes cannot. The fluorescence of Al-MOGs at 467 nm was seriously quenched by picric acid (PA) whose lowest unoccupied molecular orbital (LUMO) energy levels are lower than those of three other hydroxyl nitro aromatic compounds including 4-nitrophenol (4-NP), 3,5-dinitrosalicylic acid (3,5-DNTSA) and 2,4-dinitrophenol (2,4-DNP). Thus, PA was chosen as a model compound under optimal conditions and the relative fluorescence intensity of Al-MOGs was proportional to the concentration of PA in the range of 5.0-320.0 μM with a detection limit of 4.64 μM. Furthermore, the fluorescence quenching mechanism has also been investigated and revealed that the quenching was attributed to inner filter effects (IFEs), as well as electron transfer (ET) between Al-MOGs and PA.

Al-based metal-organic gels for selective fluorescence recognition of hydroxyl nitro aromatic compounds.

PubMed

Guo, Mao Xia; Yang, Liu; Jiang, Zhong Wei; Peng, Zhe Wei; Li, Yuan Fang

2017-12-05

The novel class of luminescent Al 3+ -based metal-organic gels (Al-MOGs) have been developed by mix 4-[2,2':6',2″-terpyridine]-4'-ylbenzoic acid (Hcptpy) with Al 3+ under mild condition. The as-prepared Al-MOGs have not only multiple stimuli-responsive properties, but selective recognition of hydroxyl nitro aromatic compounds, which can quench the fluorescence of the Al-MOGs, while other nitro aromatic analogues without hydroxyl substitutes cannot. The fluorescence of Al-MOGs at 467nm was seriously quenched by picric acid (PA) whose lowest unoccupied molecular orbital (LUMO) energy levels are lower than those of three other hydroxyl nitro aromatic compounds including 4-nitrophenol (4-NP), 3,5-dinitrosalicylic acid (3,5-DNTSA) and 2,4-dinitrophenol (2,4-DNP). Thus, PA was chosen as a model compound under optimal conditions and the relative fluorescence intensity of Al-MOGs was proportional to the concentration of PA in the range of 5.0-320.0μM with a detection limit of 4.64μM. Furthermore, the fluorescence quenching mechanism has also been investigated and revealed that the quenching was attributed to inner filter effects (IFEs), as well as electron transfer (ET) between Al-MOGs and PA. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescence kinetics of emission from a small finite volume of a biological system

NASA Astrophysics Data System (ADS)

Dagen, A. J.; Alfano, R. R.; Zilinskas, B. A.; Swenberg, C. E.

1985-07-01

The fluorescence decay, apparent quantum yield and transmission from chromophores constrained to a microscopic volume using a single picosecond laser excitation were measured as a function of incident intensity. The β subunit of phycoeryhthrin aggregate isolated from the photosynthetic antenna system of Nostoc sp. was selected since it contains only four chromophores in a volume of less than 5.6×10 4 Å 3. The non-exponential fluorescence decay profiles were intensity independent for the intensity range studied (5 × 10 13 - 2 × 10 15 photon cm -2 per pulse). The apparent decrease in the relative fluorescence quantum yield and increase of the relative transmission with increasing excitation intensity is attributed to the combined effects of ground state depletion and upper excited state absorption. Evidence suggests that exciton annihilation is absent within isolated β subunits.

Fluorescent pH sensor based on Ag@SiO2 core-shell nanoparticle.

PubMed

Bai, Zhenhua; Chen, Rui; Si, Peng; Huang, Youju; Sun, Handong; Kim, Dong-Hwan

2013-06-26

We have demonstrated a novel method for the preparation of a fluorescence-based pH sensor by combining the plasmon resonance band of Ag core and pH sensitive dye (HPTS). A thickness-variable silica shell is placed between Ag core and HPTS dye to achieve the maximum fluorescence enhancement. At the shell thickness of 8 nm, the fluorescence intensity increases 4 and 9 times when the sensor is excited at 405 and 455 nm, respectively. At the same time, the fluorescence intensity shows a good sensitivity toward pH value in the range of 5-9, and the ratio of emission intensity at 513 nm excited at 455 nm to that excited at 405 nm versus the pH value in the range of 5-9 is determined. It is believed that the present pH sensor has the potential for determining pH real time in the biological sample.

Phytoplankton-Fluorescence-Lifetime Vertical Profiler

NASA Technical Reports Server (NTRS)

Fernandez, Salvador M.; Guignon, Ernest F.; St. Louis, Ernest

2004-01-01

A battery-operated optoelectronic instrument is designed to be lowered into the ocean to measure the intensity and lifetime of fluorescence of chlorophyll A in marine phytoplankton as a function of depth from 0 to 300 m. Fluorescence lifetimes are especially useful as robust measures of photosynthetic productivity of phytoplankton and of physical and chemical mechanisms that affect photosynthesis. The knowledge of photosynthesis in phytoplankton gained by use of this and related instruments is expected to contribute to understanding of global processes that control the time-varying fluxes of carbon and associated biogenic elements in the ocean. The concentration of chlorophyll in the ocean presents a major detection challenge because in order to obtain accurate values of photosynthetic parameters, the intensity of light used to excite fluorescence must be kept very low so as not to disturb the photosynthetic system. Several innovations in fluorometric instrumentation were made in order to make it possible to reach the required low detection limit. These innovations include a highly efficient optical assembly with an integrated flow-through sample interface, and a high-gain, low-noise electronic detection subsystem. The instrument also incorporates means for self-calibration during operation, and electronic hardware and software for control, acquisition and analysis of data, and communications. The electronic circuitry is highly miniaturized and designed to minimize power demand. The instrument is housed in a package that can withstand the water pressure at the maximum depth of 300 m. A light-emitting diode excites fluorescence in the sample flow cell, which is placed at one focal point of an ellipsoidal reflector. A photomultiplier tube is placed at the other focal point. This optical arrangement enables highly efficient collection of fluorescence emitted over all polar directions. Fluorescence lifetime is measured indirectly, by use of a technique based on the

Polythiophene nanofilms for sensitive fluorescence detection of viruses in drinking water.

PubMed

Wankar, Shashwati; Turner, Nicholas W; Krupadam, Reddithota J

2016-08-15

Molecular imprints of the tobacco necrosis virus (TNV) have been formed within polythiophene nanofilms with an approximate thickness of 200nm. These films have been electrochemically deposited onto conducting Au surfaces. Upon rebinding, the TNV-polythiophene complex changes the fluorescence intensity of the nanofilm. The fluorescence intensity at 410nm was observed to be proportional to the concentration of viruses in the range of 0.1-10ngL(-1) (0.15-15pg) with the lower calculated detection limit of 2.29ngL(-1) (3.4pg). The intensity of the fluorescence emission is not affected by the thickness of the polythiophene film and the nature of TNV specific binding sites. Kinetic data analyses showed that the nanofilm responds to TNV within 2min; and cross-selectivity studies with tobacco mosaic virus (TMV) showed an excellent specificity for the targeted TNV. These binding experiments demonstrate the potential of fluorescence emission for the specific, label free and rapid detection of viruses using nanofilm sensors. Taking into account the lower limit of detection, the fluorescence sensing reported here is reliable, simple to perform, rapid, cost-effective and offers a sensitive analytical method for virus detection in water resources. Copyright © 2016 Elsevier B.V. All rights reserved.

Optical properties of flexible fluorescent films prepared by screen printing technology

NASA Astrophysics Data System (ADS)

Chen, Yan; Ke, Taiyan; Chen, Shuijin; He, Xin; Zhang, Mei; Li, Dong; Deng, Jinfeng; Zeng, Qingguang

2018-05-01

In this work, we prepared a fluorescent film comprised phosphors and silicone on flexible polyethylene terephthalate (PET) substrate using a screen printing technology. The effects of mesh number and weight ratio of phosphors to silicone on the optical properties of the flexible films were investigated. The results indicate that the emission intensity of the film increase as the mesh decreased from 400 to 200, but the film surface gradually becomes uneven. The fluorescent film with high emission intensity and smooth surface can be obtained when the weight ratio of phosphor to gel is 2:1, and mesh number is 300. The luminous efficiency of the fabricated LEDs combined the fluorescent films with 460 nm Ga(In)N chip module can reach 75 lm/W. The investigation indicates that the approach can be applied in the remote fluorescent film conversion and decreases the requirements of the particle size and the dispersion state of fluorescent materials.

Preliminary study on X-ray fluorescence computed tomography imaging of gold nanoparticles: Acceleration of data acquisition by multiple pinholes scheme

NASA Astrophysics Data System (ADS)

Sasaya, Tenta; Sunaguchi, Naoki; Seo, Seung-Jum; Hyodo, Kazuyuki; Zeniya, Tsutomu; Kim, Jong-Ki; Yuasa, Tetsuya

2018-04-01

Gold nanoparticles (GNPs) have recently attracted attention in nanomedicine as novel contrast agents for cancer imaging. A decisive tomographic imaging technique has not yet been established to depict the 3-D distribution of GNPs in an object. An imaging technique known as pinhole-based X-ray fluorescence computed tomography (XFCT) is a promising method that can be used to reconstruct the distribution of GNPs from the X-ray fluorescence emitted by GNPs. We address the acceleration of data acquisition in pinhole-based XFCT for preclinical use using a multiple pinhole scheme. In this scheme, multiple projections are simultaneously acquired through a multi-pinhole collimator with a 2-D detector and full-field volumetric beam to enhance the signal-to-noise ratio of the projections; this enables fast data acquisition. To demonstrate the efficacy of this method, we performed an imaging experiment using a physical phantom with an actual multi-pinhole XFCT system that was constructed using the beamline AR-NE7A at KEK. The preliminary study showed that the multi-pinhole XFCT achieved a data acquisition time of 20 min at a theoretical detection limit of approximately 0.1 Au mg/ml and at a spatial resolution of 0.4 mm.

Detection of rheumatoid arthritis in humans by fluorescence imaging

NASA Astrophysics Data System (ADS)

Ebert, Bernd; Dziekan, Thomas; Weissbach, Carmen; Mahler, Marianne; Schirner, Michael; Berliner, Birgitt; Bauer, Daniel; Voigt, Jan; Berliner, Michael; Bahner, Malte L.; Macdonald, Rainer

2010-02-01

The blood pool agent indo-cyanine green (ICG) has been investigated in a prospective clinical study for detection of rheumatoid arthritis using fluorescence imaging. Temporal behavior as well as spatial distribution of fluorescence intensity are suited to differentiate healthy and inflamed finger joints after i.v. injection of an ICG bolus.

Nanostructures Derived from Starch and Chitosan for Fluorescence Bio-Imaging

PubMed Central

Zu, Yinxue; Bi, Jingran; Yan, Huiping; Wang, Haitao; Song, Yukun; Zhu, Bei-Wei; Tan, Mingqian

2016-01-01

Fluorescent nanostructures (NSs) derived from polysaccharides have drawn great attention as novel fluorescent probes for potential bio-imaging applications. Herein, we reported a facile alkali-assisted hydrothermal method to fabricate polysaccharide NSs using starch and chitosan as raw materials. Transmission electron microscopy (TEM) demonstrated that the average particle sizes are 14 nm and 75 nm for starch and chitosan NSs, respectively. Fourier transform infrared (FT-IR) spectroscopy analysis showed that there are a large number of hydroxyl or amino groups on the surface of these polysaccharide-based NSs. Strong fluorescence with an excitation-dependent emission behaviour was observed under ultraviolet excitation. Interestingly, the photostability of the NSs was found to be superior to fluorescein and rhodamine B. The quantum yield of starch NSs could reach 11.12% under the excitation of 360 nm. The oxidative metal ions including Cu(II), Hg(II)and Fe(III) exhibited a quench effect on the fluorescence intensity of the prepared NSs. Both of the two kinds of the multicoloured NSs showed a maximum fluorescence intensity at pH 7, while the fluorescence intensity decreased dramatically when they were put in an either acidic or basic environment (at pH 3 or 11). The cytotoxicity study of starch NSs showed that low cell cytotoxicity and 80% viability was found after 24 h incubation, when their concentration was less than 10 mg/mL. The study also showed the possibility of using the multicoloured starch NSs for mouse melanoma cells and guppy fish imaging. PMID:28335258

Fluorescence spectral properties of stomach tissues with pathology

NASA Astrophysics Data System (ADS)

Giraev, K. M.; Ashurbekov, N. A.; Lahina, M. A.

2012-05-01

Steady-state fluorescence and diffuse reflection spectra are measured for in vivo normal and pathological (chronic atrophic and ulcerating defects, malignant neoplasms) stomach mucous lining tissues. The degree of distortion of the fluorescence spectra is estimated taking light scattering and absorption into account. A combination of Gauss and Lorentz functions is used to decompose the fluorescence spectra. Potential groups of fluorophores are determined and indices are introduced to characterize the dynamics of their contributions to the resultant spectra as pathologies develop. Reabsorption is found to quench the fluorescence of structural proteins by as much as a factor of 3, while scattering of the light can increase the fluorescence intensity of flavin and prophyrin groups by as much as a factor of 2.

Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection

PubMed Central

Bates, Mark; Dempsey, Graham T; Chen, Kok Hao; Zhuang, Xiaowei

2012-01-01

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution. PMID:22213647

Photostability effect of silica nanoparticles encapsulated fluorescence dye

NASA Astrophysics Data System (ADS)

Ahmad, Atiqah; Zakaria, Nor Dyana; Razak, Khairunisak Abdul

2017-12-01

Fluorescence dyes are based on small organic molecules have become of interest in chemical biology and widely used for cell and intracellular imaging. However, fluorescence dyes have limitations such as photo bleaching, poor photochemical stability and has a short Stokes shift. It is less valuable for long-term cell tracking strategies and has very short lifetime. In order to overcome the problems, dye-incorporated nanomaterials become of interest. Nanomaterials encapsulation provides a protection layer around the fluorescence dye which improves the stability of fluorescence dye. In this study, silica nanoparticles encapsulated with 1,1%-dioctadecyl-3,3,3%,3%-tetramethylindocarbocyanine perchlorate (Dil) was successfully synthesised by using micelle entrapment method to investigate the effect of encapsulation of nanoparticles towards the properties of fluorescent dye. The synthesised nanoparticles (SiDil) was characterised by particle size analyser, Transmission Electron Microscopy (TEM), UV-Vis spectrometer and Fluorescent spectrometer. Observation using TEM showed spherical shape of nanoparticles with 53 nm diameter. Monodispersed and well nanoparticles distribution was confirmed by low polydispersity index of 0.063 obtained by particle size analyser. Furthermore, the photoluminescence properties of the SiDil were evaluated and compared with bare Dil dye. Both SiDil and bare Dil was radiated under 200 W of Halogen lamp for 60 minutes and the absorbance intensity was measured using UV-Vis spectrometer. The result showed more stable absorbance intensity for SiDil compared to bare Dil dye, which indicated that Si nanoparticles encapsulation improved the photostability property.

Effect of surfactant and budesonide on the pulmonary distribution of fluorescent dye in mice.

PubMed

Huang, Liang-Ti; Yeh, Tsu-Fu; Kuo, Yu-Lin; Chen, Pin-Chuan; Chen, Chung-Ming

2015-02-01

Surfactant is a useful vehicle for the intratracheal delivery of medicine to the distal lung. The aim of this study was to analyze the effect of intratracheal surfactant and budesonide instillation on the pulmonary distribution of fluorescent dye in mice. Male athymic nude mice were assigned randomly as controls, fluorescent dye, fluorescent dye + surfactant (50 mg/kg), fluorescent dye + budesonide (0.25 mg/kg), and fluorescent dye + surfactant + budesonide groups. A total volume of 60 μL fluorescent solutions was intratracheally injected and followed by 60 μL of air. We photographed and measured fluorescence in the lungs, from the back, 15 minutes after intratracheal administration using an IVIS Xenogen imaging instrument. The fluorescent dye (1,1'-dioctadecyltetramethyl indotricarbocyanine iodide) was most strongly detected near the trachea and weakly detected in the lungs in mice administered with fluorescent solutions. Almost no fluorescence was seen in the lung region of control mice. Intratracheal administration of surfactant or budesonide increased fluorescent intensity compared with control mice. Combined administration of surfactant and budesonide further increased fluorescent intensity compared with mice given surfactant or budesonide alone. Surfactant and budesonide enhance the pulmonary distribution of fluorescent dye in mice. Copyright © 2014. Published by Elsevier B.V.

Intensive exposure as a risk factor for severe polio: a study of multiple family cases.

PubMed

Nielsen, N M; Aaby, P; Wohlfahrt, J; Pedersen, J B; Melbye, M; Mølbak, K

2001-01-01

To examine the importance of intensity of exposure for the outcome of the poliomyelitis infection 429 polio cases were identified belonging to families with 2, 3 or 4 polio cases, all hospitalized in Copenhagen from 1919 to 1953. Furthermore, 87 pairs of polio cases living on the same stairway, but not in the same household, were identified. Severity among multiple cases in families analysed according to time of appearance showed a U-shaped curve. Initial cases had a higher risk of developing paralysis [relative risk (RR) = 1.5, 95% confidence interval (CI) 1.2-1.91 and of dying (RR = 2.5, 95% CI 0.9-6.9). Decreased severity was observed among subsequent cases appearing within 11 d after the initial case (RR = 1.0); however, severity increased again, with higher mortality for cases likely to have been infected by the initial case (cases appearing more than 11 d later) (RR = 5.7, 95% CI 1.8-17.8). The pattern described among multiple family cases was not found among cases from the same stairway. Since family cases appearing within 11 d were probably infected simultaneously, a short incubation period is associated with severe disease and a prolonged incubation period with milder infections. Furthermore, intensive exposure from being infected in the household increased severity. These observations therefore suggest that intensity of exposure and dose of infection are important factors in the severity of poliomyelitis.

Polarization of fluorescein fluorescence in single cells.

PubMed

Udkoff, R; Norman, A

1979-01-01

Measurement of fluorescence polarization (P) gives information about the immediate environment of the fluorescent molecule. We used a flow polarimeter to investigate the factors influencing P of fluorescein in mammalian cells to determine whether such measurements are useful for characterizing heterogeneous cell populations. Fluorescein was introduced into cells by incubation with FDA. Measurements of the intensity of fluorescence (TI) and polarization (P) revealed an unexpected dependence: P decreased with increasing intensity of fluorescence. This may be accounted for by the classical model of the binding of small molecules to protein in which P is dependent on the ratio bound to unbound molecules. We have been able to estimate the quenching due to binding and construct a Scatchard plot. We estimated a wavelength shift from in vitro data consistent with the dependence of P on wavelength seen in our cell work. Generally, the distributions of P are symmetrical. Photon statistics broadens the P distribution of dim cells. However, structure does develop in the P distribution when the cells are deprived of calcium or incubated in the cold. This appears as a shoulder on the P distribution or resolves into two peaks. Calcium deprivation may differentially affect a subpopulation of cells whose significance remains to be explored in various cell types.

Robust detection of multiple sclerosis lesions from intensity-normalized multi-channel MRI

NASA Astrophysics Data System (ADS)

Karpate, Yogesh; Commowick, Olivier; Barillot, Christian

2015-03-01

Multiple sclerosis (MS) is a disease with heterogeneous evolution among the patients. Quantitative analysis of longitudinal Magnetic Resonance Images (MRI) provides a spatial analysis of the brain tissues which may lead to the discovery of biomarkers of disease evolution. Better understanding of the disease will lead to a better discovery of pathogenic mechanisms, allowing for patient-adapted therapeutic strategies. To characterize MS lesions, we propose a novel paradigm to detect white matter lesions based on a statistical framework. It aims at studying the benefits of using multi-channel MRI to detect statistically significant differences between each individual MS patient and a database of control subjects. This framework consists in two components. First, intensity standardization is conducted to minimize the inter-subject intensity difference arising from variability of the acquisition process and different scanners. The intensity normalization maps parameters obtained using a robust Gaussian Mixture Model (GMM) estimation not affected by the presence of MS lesions. The second part studies the comparison of multi-channel MRI of MS patients with respect to an atlas built from the control subjects, thereby allowing us to look for differences in normal appearing white matter, in and around the lesions of each patient. Experimental results demonstrate that our technique accurately detects significant differences in lesions consequently improving the results of MS lesion detection.

Fast Decomposition of Three-Component Spectra of Fluorescence Quenching by White and Grey Methods of Data Modeling.

PubMed

Kałka, Andrzej J; Turek, Andrzej M

2018-04-03

'White' and 'grey' methods of data modeling have been employed to resolve the heterogeneous fluorescence from a fluorophore mixture of 9-cyanoanthracene (CNA), 10-chloro-9-cyanoanthracene (ClCNA) and 9,10-dicyanoanthracene (DCNA) into component individual fluorescence spectra. The three-component spectra of fluorescence quenching in methanol were recorded for increasing amounts of lithium bromide used as a quencher. The associated intensity decay profiles of differentially quenched fluorescence of single components were modeled on the basis of a linear Stern-Volmer plot. These profiles are necessary to initiate the fitting procedure in both 'white' and 'grey' modeling of the original data matrices. 'White' methods of data modeling, called also 'hard' methods, are based on chemical/physical laws expressed in terms of some well-known or generally accepted mathematical equations. The parameters of these models are not known and they are estimated by least squares curve fitting. 'Grey' approaches to data modeling, also known as hard-soft modeling techniques, make use of both hard-model and soft-model parts. In practice, the difference between 'white' and 'grey' methods lies in the way in which the 'crude' fluorescence intensity decays of the mixture components are estimated. In the former case they are given in a functional form while in the latter as digitized curves which, in general, can only be obtained by using dedicated techniques of factor analysis. In the paper, the initial values of the Stern-Volmer constants of pure components were evaluated by both 'point-by-point' and 'matrix' versions of the method making use of the concept of wavelength dependent intensity fractions as well as by the rank annihilation factor analysis applied to the data matrices of the difference fluorescence spectra constructed in two ways: from the spectra recorded for a few excitation lines at the same concentration of a fluorescence quencher or classically from a series of the spectra

Metal-enhanced fluorescence/visual bimodal platform for multiplexed ultrasensitive detection of microRNA with reusable paper analytical devices.

PubMed

Liang, Linlin; Lan, Feifei; Yin, Xuemei; Ge, Shenguang; Yu, Jinghua; Yan, Mei

2017-09-15

Convenient biosensor for simultaneous multi-analyte detection was increasingly required in biological analysis. A novel flower-like silver (FLS)-enhanced fluorescence/visual bimodal platform for the ultrasensitive detection of multiple miRNAs was successfully constructed for the first time based on the principle of multi-channel microfluidic paper-based analytical devices (µPADs). Fluorophore-functionalized DNA 1 (DNA 1 -N-CDs) was combined with FLS, which was hybridized with quencher-carrying strand (DNA 2 -CeO 2 ) to form FLS-enhanced fluorescence biosensor. Upon the addition of the target miRNA, the fluorescent intensity of DNA 1 -N-CDs within the proximity of the FLS was strengthened. The disengaged DNA/CeO 2 complex could result in color change after joining H 2 O 2 , leading to real-time visual detection of miRNA firstly. If necessary, then the fluorescence method was applied for a accurate determination. In this strategy, the growth of FLS in µPADs not only reduced the background fluorescence but also provided an enrichment of "hot spots" for surface enhanced fluorescence detection of miRNAs. Results also showed versatility of the FLS in the enhancement of sensitivity and selectivity of the miRNA biosensor. Remarkably, this biosensor could detect as low as 0.03fM miRNA210 and 0.06fM miRNA21. Interestingly, the proposed biosensor also possessed good capability of recycling in three cycles upon change of the supplementation of DNA 2 -CeO 2 and visual substitutive device. This method opened new opportunities for further studies of miRNA related bioprocesses and will provide a new instrument for simultaneous detection of multiple low-level biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

The fluorescence properties of aerosol larger than 0.8 μm in an urban and a PBA-dominated location

NASA Astrophysics Data System (ADS)

Gabey, A. M.; Stanley, W. R.; Gallagher, M. W.; Kaye, P. H.

2011-01-01

Dual-wavelength Ultraviolet light-induced fluorescence (UV-LIF) measurements were performed on ambient environmental aerosol in Manchester, UK (urban city centre, winter) and Borneo, Malaysia (remote, tropical), which are taken to represent environments with negligible and significant primary biological aerosol (PBA) influences, respectively. Single-particle fluorescence intensity and optical equivalent diameter were measured with a Wide Issue Bioaerosol Sensor, version 3 (WIBS3) in the diameter range 0.8 μm≤DP≤20 μm for 2-3 weeks and filters were analysed using energy dispersive X-ray (EDX) spectroscopy, which revealed mostly non-PBA dominated particle sizes larger than 1 μm in Manchester. The WIBS3 features three fluorescence channels: Fluorescence excited at 280 nm is recorded at 310-400 nm and 400-600 nm and fluorescence excited at 370 nm is detected at 400-600 nm. In Manchester the primary size mode of fluorescent and non-fluorescent material was at 1.2 μm. In Borneo non-fluorescent material peaked at 1.2 μm and fluorescent at 3-4 μm. The fluorescence intensity at 400-600 nm generally increased with DP at both sites, as did the 310-400 nm intensity in Borneo. In Manchester the 310-400 m fluorescence decreased at DP>4 μm, suggesting this channel offers additional discrimination between fluorescent particle types. Finally, the ratio of fluorescence intensity in two pairs of channels was investigated as a function of particle diameter and this varied significantly between the two environments, demonstrating that the fluorescent aerosol in each can in principle be distinguished using a combination of fluorescence and elastic scattering measurements.

Fluorescence image excited by a scanning UV-LED light

NASA Astrophysics Data System (ADS)

Tsai, Hsin-Yi; Chen, Yi-Ju; Huang, Kuo-Cheng

2013-03-01

An optical scanning system using UV-LED light to induced fluorescence technology can enhance a fluorescence image significantly in a short period. It has several advantages such as lower power consumption, no scattering effect in skins, and multilayer images can be obtained to analyze skin disease. From the experiment results, the light intensity increases with increase spot size and decrease scanning speed, but the image resolution is oppositely. Moreover, the system could be widely used in clinical diagnosis and photodynamic therapy for skin disease because even the irradiated time of fluorescence substance is short but it will provide accurately positioning of fluorescence object.

Fluorescence-based enhanced reality (FLER) for real-time estimation of bowel perfusion in minimally invasive surgery

NASA Astrophysics Data System (ADS)

Diana, Michele

2016-03-01

Pre-anastomotic bowel perfusion is a key factor for a successful healing process. Clinical judgment has limited accuracy to evaluate intestinal microperfusion. Fluorescence videography is a promising tool for image-guided intraoperative assessment of the bowel perfusion at the future anastomotic site in the setting of minimally invasive procedures. The standard configuration for fluorescence videography includes a Near-Infrared endoscope able to detect the signal emitted by a fluorescent dye, more frequently Indocyanine Green (ICG), which is administered by intravenous injection. Fluorescence intensity is proportional to the amount of fluorescent dye diffusing in the tissue and consequently is a surrogate marker of tissue perfusion. However, fluorescence intensity alone remains a subjective approach and an integrated computer-based analysis of the over-time evolution of the fluorescence signal is required to obtain quantitative data. We have developed a solution integrating computer-based analysis for intra-operative evaluation of the optimal resection site, based on the bowel perfusion as determined by the dynamic fluorescence intensity. The software can generate a "virtual perfusion cartography", based on the "fluorescence time-to-peak". The virtual perfusion cartography can be overlapped onto real-time laparoscopic images to obtain the Enhanced Reality effect. We have defined this approach FLuorescence-based Enhanced Reality (FLER). This manuscript describes the stepwise development of the FLER concept.

The fluorescent photobleaching properties of GFP expressed in human lung cancer cells

NASA Astrophysics Data System (ADS)

Jin, Ying; Xing, Da

2003-12-01

The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the dicistronic expression vector (pEGFP-C1) was used to transfected into human lung cancer cell line (ASTC-a-1) and a positive clone which stably expressed GFP in high level was obtained. After more than three months' passengers, the cells were also remained the strong fluorescence under fluorescent microscope. The results showed that the green fluorescent protein expressed in tumor cells was also photobleached under intense irradiation (approximately 488 nm) and the degree of photobleaching varied with the difference of the intensity of the excitation. Using different interdiction parcel (None, ND4, ND8, ND16), there were significant differences in photobleaching among the different excitation. The photobleaching was also affected by the time length of excitation, and the intensity of fluorescence was obviously decreased along with the increasing of excitation time, especially to stronger excitation.

Micro-Raman spectroscopy of algae: composition analysis and fluorescence background behavior.

PubMed

Huang, Y Y; Beal, C M; Cai, W W; Ruoff, R S; Terentjev, E M

2010-04-01

Preliminary feasibility studies were performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella sorokiniana (UTEX #1230) and Neochloris oleoabundans (UTEX #1185), were chosen for this study. Both species were considered to be candidates for biofuel production. Raman signals due to storage lipids (specifically triglycerides) were clearly identified in the nitrogen-starved C. sorokiniana and N. oleoabundans, but not in their healthy counterparts. On the other hand, signals resulting from the carotenoids were found to be present in all of the samples. Composition mapping was conducted in which Raman spectra were acquired from a dense sequence of locations over a small region of interest. The spectra obtained for the mapping images were filtered for the wavelengths of characteristic peaks that correspond to components of interest (i.e., triglyceride or carotenoid). The locations of the components of interest could be identified by the high intensity areas in the composition maps. Finally, the time evolution of fluorescence background was observed while acquiring Raman signals from the algae. The time dependence of fluorescence background is characterized by a general power law decay interrupted by sudden high intensity fluorescence events. The decreasing trend is likely a result of photo-bleaching of cell pigments due to prolonged intense laser exposure, while the sudden high intensity fluorescence events are not understood. (c) 2009 Wiley Periodicals, Inc.

Near-infrared squaraine dyes for fluorescence enhanced surface assay

PubMed Central

Matveeva, Evgenia G.; Terpetschnig, Ewald A.; Stevens, Megan; Patsenker, Leonid; Kolosova, Olga S.; Gryczynski, Zygmunt; Gryczynski, Ignacy

2009-01-01

Commercially available, near-infrared fluorescent squaraine dyes (Seta-635 and Seta-670) were covalently bound to antibodies and employed insurface enhanced immunoassay. From fluorescence intensity and lifetime changes determined for a surface which had been coated with silver nanoparticles as well as a non-coated glass surface, both labelled compounds exhibited a 15 to 20-fold enhancement of fluorescence on the silver coated surface compared to that achieved on the non-coated surface. In addition, the fluorescence lifetime changes drastically for both labels in the case of silver-coated surfaces. The fluorescence signal enhancement obtained for the two dyes was greater than that previously recorded for Rhodamine Red-X and AlexaFluor-647 labels. PMID:20046935

Red-emitting fluorescent probe for detecting hypochlorite acid in vitro and in vivo.

PubMed

Chen, Hong; Sun, Tao; Qiao, Xiao-Guang; Tang, Qian-Oian; Zhao, Shan-Chao; Zhou, Zhan

2018-06-12

Due to the importance of hypochlorous acid (HClO) in biological and industrial, development of fluorescent probes for HClO has been an active research area. Here, a new red-emitting ratiometric fluorescent probe (P) was synthesized and well defined characterization via NMR, HR-MS, and fluorescence spectrum, which serves as a selective and sensitive probe for ClO - group. The probe showed a ratiometric fluorescent response to hypochlorite at the emission intensities ratio (I 480 /I 612 ) increasing from 0.28 to 27.46. The emission intensities ratio (I 480 /I 612 ) was linearly enhanced (I 480 /I 612  = 0.064 X + 0.096) with the ClO - concentration range from 1 to 30 μM. The detection limitation for ClO - in aqueous solution is 0.47 μM. Moreover, this biocompatible red-emitting ratiometric fluorescent probe was utilized to the fluorescence imaging of ClO - in living cells and Zebrafish. Copyright © 2018. Published by Elsevier B.V.

Arthritis imaging using a near-infrared fluorescence folate-targeted probe

PubMed Central

Chen, Wei-Tsung; Mahmood, Umar; Weissleder, Ralph; Tung, Ching-Hsuan

2005-01-01

A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate (n = 12) and of free NIR2 (n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model (n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases. PMID:15743478

Algorithms for differentiating between images of heterogeneous tissue across fluorescence microscopes.

PubMed

Chitalia, Rhea; Mueller, Jenna; Fu, Henry L; Whitley, Melodi Javid; Kirsch, David G; Brown, J Quincy; Willett, Rebecca; Ramanujam, Nimmi

2016-09-01

Fluorescence microscopy can be used to acquire real-time images of tissue morphology and with appropriate algorithms can rapidly quantify features associated with disease. The objective of this study was to assess the ability of various segmentation algorithms to isolate fluorescent positive features (FPFs) in heterogeneous images and identify an approach that can be used across multiple fluorescence microscopes with minimal tuning between systems. Specifically, we show a variety of image segmentation algorithms applied to images of stained tumor and muscle tissue acquired with 3 different fluorescence microscopes. Results indicate that a technique called maximally stable extremal regions followed by thresholding (MSER + Binary) yielded the greatest contrast in FPF density between tumor and muscle images across multiple microscopy systems.

Combining OCT and a fluorescence intensity imaging method for atherosclerosis detection

NASA Astrophysics Data System (ADS)

Liang, Shanshan; Saidi, Arya; Jing, Joe; Liu, Gangjun; Yin, Jiechen; Narula, Jagat; Chen, Zhongping

2012-02-01

Coronary heart disease (like myocardial infarction) is caused by atherosclerosis. It cause over 30% of all deaths in North America and are the most common cause of death in European men under 65 years of age and the second most common cause in women. To diagnose this atherosclerosis before it gets rupture is the most effect way to increase the chance of survival for patients who suffer from this disease. The crucial tusk is how to find out vulnerable plaques. In resent years optical coherence tomography (OCT) has become a very useful tool for intravascular imaging, since it has high axial and transverse resolution. OCT can tell the detail structure inside the plaque like the thickness of plaque cap which is an important factor to identify vulnerable plaques. But we still need to find out the biochemical characteristics that is unique for vulnerable plaques (like inflammation). Fluorescence molecular imaging is a standard way to exam the biochemical property of biological samples. So we integrate these two techniques together into one probe. Our probe is comprised of a double-clad fiber (DCF) and a grin lens, and rotates with a micro mirror in front. The single-mode inner core of the DCF transmits both OCT and fluorescence excitation light, and the multimode inner cladding is used to detect fluorescence signal. In vitro result shows that this is a possible way for more accurate diagnose of vulnerable plaques.

Preassembled Fluorescent Multivalent Probes for the Imaging of Anionic Membranes.

PubMed

Roland, Felicia M; Peck, Evan M; Rice, Douglas R; Smith, Bradley D

2017-04-19

A new self-assembly process known as Synthavidin (synthetic avidin) technology was used to prepare targeted probes for near-infrared fluorescence imaging of anionic membranes and cell surfaces, a hallmark of many different types of disease. The probes were preassembled by threading a tetralactam macrocycle with six appended zinc-dipicolylamine (ZnDPA) targeting units onto a linear scaffold with one or two squaraine docking stations to produce hexavalent or dodecavalent fluorescent probes. A series of liposome titration experiments showed that multivalency promoted stronger membrane binding by the dodecavalent probe. In addition, the dodecavalent probe exhibited turn-on fluorescence due to probe unfolding during fluorescence microscopy at the membrane surface. However, the dodecavalent probe also had a higher tendency to self-aggregate after membrane binding, leading to probe self-quenching under certain conditions. This self-quenching effect was apparent during fluorescence microscopy experiments that recorded low fluorescence intensity from anionic dead and dying mammalian cells that were saturated with the dodecavalent probe. Conversely, probe self-quenching was not a factor with anionic microbial surfaces, where there was intense fluorescence staining by the dodecavalent probe. A successful set of rat tumor imaging experiments confirmed that the preassembled probes have sufficient mechanical stability for effective in vivo imaging. The results demonstrate the feasibility of this general class of preassembled fluorescent probes for multivalent targeting, but fluorescence imaging performance depends on the specific physical attributes of the biomarker target, such as the spatial distance between different copies of the biomarker and the propensity of the probe-biomarker complex to self-aggregate.

Calibration of Wide-Field Deconvolution Microscopy for Quantitative Fluorescence Imaging

PubMed Central

Lee, Ji-Sook; Wee, Tse-Luen (Erika); Brown, Claire M.

2014-01-01

Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure. PMID:24688321

Measurement of the fluorescence of crop residues: A tool for controlling soil erosion

NASA Technical Reports Server (NTRS)

Daughtry, C. S. T.; Mcmurtrey, J. E., III; Chappelle, E. W.; Hunter, W. J.

1994-01-01

Management of crop residues, the portion of a crop left in the field after harvest, is an important conservation practice for minimizing soil erosion and for improving water quality. Quantification of crop residue cover is required to evaluate the effectiveness of conservation tillage practices. Methods are needed to quantify residue cover that are rapid, accurate, and objective. The fluorescence of crop residue was found to be a broadband phenomenon with emission maxima at 420 to 495 nm for excitations of 350 to 420 nm. Soils had low intensity broadband emissions over the 400 to 690 nm region for excitations of 300 to 600 nm. The range of relative fluorescence intensities for the crop residues was much greater than the fluorescence observed of the soils. As the crop residues decompose their blue fluorescence values approach the fluorescence of the soil. Fluorescence techniques are concluded to be less ambiguous and better suited for discriminating crop residues and soils than reflectance methods. If properly implemented, fluorescence techniques can be used to quantify, not only crop residue cover, but also photosynthetic efficiency in the field.

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

NASA Astrophysics Data System (ADS)

Bogh, Sidsel A.; Bora, Ilkay; Rosenberg, Martin; Thyrhaug, Erling; Laursen, Bo W.; Just Sørensen, Thomas

2015-12-01

Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r 0  =  0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (ϕ fl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕ fl, a ~20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.

A novel small molecule mediate 18F-FDG excited fluorescence molecular imaging

NASA Astrophysics Data System (ADS)

Zhang, Zeyu; Guo, Hongbo; Hu, Zhenhua; Tian, Jie

2018-02-01

Fluorescence molecular imaging (FMI) has been widely used in many medical fields with small molecule indocyanine green (ICG). However, low signal-background ratio and limited specificity to tumor remain big challenges for FMI. In this study, a novel excitation strategy is proposed on the basis of clinical approved ICG and 18F-FDG. A series of in vitro experiments are designed to reveal the mechanism and results show obvious decreasing of ICG fluorescence intensity with the increasing distance to excitation source. Meanwhile, the ICG fluorescence intensity is proportional to the activity of radiopharmaceutical. Results from different respects illustrate the promising of this proposed excitation strategy.

Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions

NASA Astrophysics Data System (ADS)

Sharifzadeh, Mohsen; Bernstein, Paul S.; Gellermann, Werner

2006-10-01

We have developed a CCD-camera-based nonmydriatic instrument that detects fluorescence from retinal lipofuscin chromophores ("autofluorescence") as a means to indirectly quantify and spatially image the distribution of macular pigment (MP). The lipofuscin fluorescence intensity is reduced at all retinal locations containing MP, since MP has a competing absorption in the blue-green wavelength region. Projecting a large diameter, 488 nm excitation spot onto the retina, centered on the fovea, but extending into the macular periphery, and comparing lipofuscin fluorescence intensities outside and inside the foveal area, it is possible to spatially map out the distribution of MP. Spectrally selective detection of the lipofuscin fluorescence reveals an important wavelength dependence of the obtainable image contrast and deduced MP optical density levels, showing that it is important to block out interfering fluorescence contributions in the detection setup originating from ocular media such as the lens. Measuring 70 healthy human volunteer subjects with no ocular pathologies, we find widely varying spatial extent of MP, distinctly differing distribution patterns of MP, and strongly differing absolute MP levels among individuals. Our population study suggests that MP imaging based on lipofuscin fluorescence is useful as a relatively simple, objective, and quantitative noninvasive optical technique suitable to rapidly screen MP levels and distributions in healthy humans with undilated pupils.

A compact multi-channel fluorescence sensor with ambient light suppression

NASA Astrophysics Data System (ADS)

Egly, Dominik; Geörg, Daniel; Rädle, Matthias; Beuermann, Thomas

2012-03-01

A multi-channel fluorescence sensor has been developed for process monitoring and fluorescence diagnostics. It comprises a fiber-optic set-up with an immersion probe and an intensity-modulated high power ultraviolet light-emitting diode as a light source for fluorescence excitation. By applying an electronic lock-in procedure, fluorescence signals are selectively detectable at ambient light levels of 1000 000 times higher intensity. The sensor was designed to be compact, low cost and easily adaptable to a wide field of application. The set-up was used to simultaneously monitor three important metabolic fluorophores: NAD(P)H, flavins and porphyrins during the cultivation of a baker's yeast. Moreover, the accumulation and degradation kinetics of protoporphyrin IX induced by 5-aminolevulinic acid on the skin could be recorded by the sensor. The detection limit for protoporphyrin IX was determined to be 4 × 10-11 mol L-1. The linear signal amplification of the sensor and time courses of fluorescence signals monitored during yeast fermentations were validated using a commercial CCD spectrometer. The robust and flexible set-up of the fiber-optic measurement system promises easy implementation of this non-invasive analytical tool to fluorescence monitoring and diagnostics in R&D and production.

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

NASA Astrophysics Data System (ADS)

Rolinski, Olaf J.; McLaughlin, Damien; Birch, David J. S.; Vyshemirsky, Vladislav

2016-09-01

The fluorescence intensity decay of protein is easily measurable and reports on the intrinsic fluorophore-local environment interactions on the sub-nm spatial and sub-ns temporal scales, which are consistent with protein activity in numerous biomedical and industrial processes. This makes time-resolved fluorescence a perfect tool for understanding, monitoring and controlling these processes at the molecular level, but the complexity of the decay, which has been traditionally fitted to multi-exponential functions, has hampered the development of this technique over the last few decades. Using the example of tryptophan in HSA we present the alternative to the conventional approach to modelling intrinsic florescence intensity decay in protein where the key factors determining fluorescence decay, i.e. the excited-state depopulation and the dielectric relaxation (Toptygin and Brand 2000 Chem. Phys. Lett. 322 496-502), are represented by the individual relaxation functions. This allows quantification of both effects separately by determining their parameters from the global analysis of a series of fluorescence intensity decays measured at different detection wavelengths. Moreover, certain pairs of the recovered parameters of tryptophan were found to be correlated, indicating the influence of the dielectric relaxation on the transient rate of the electronic transitions. In this context the potential for the dual excited state depopulation /dielectric relaxation fluorescence lifetime sensing is discussed.

Fluorescence of fungi in superficial and deep fungal infections

PubMed Central

Elston, Dirk M

2001-01-01

Background Fluorescence of many fungi is noted when H&E stained sections are examined under a fluorescent microscope. In theory, this phenomenon could aid in the diagnosis of cutaneous and disseminated fungal infections without the delay associated with special stains. Seventy-six cases of superficial and deep fungal infections and 3 cases of protothecosis were studied to determine the clinical usefulness of this technique. Results In most cases, fluorescence was noted, but was not intense. Fluorescence of fungi did not correlate with the age of the specimen. In most cases, organisms in H&E stained sections were more easily identified with routine light microscopy than with fluorescent microscopy. Conclusion This report suggests that in H&E stained skin specimens, fluorescent microscopy is of little benefit in the identification of fungal organisms. PMID:11602016

Multiple ionization of neon by soft x-rays at ultrahigh intensity

NASA Astrophysics Data System (ADS)

Guichard, R.; Richter, M.; Rost, J.-M.; Saalmann, U.; Sorokin, A. A.; Tiedtke, K.

2013-08-01

At the free-electron laser FLASH, multiple ionization of neon atoms was quantitatively investigated at photon energies of 93.0 and 90.5 eV. For ion charge states up to 6+, we compare the respective absolute photoionization yields with results from a minimal model and an elaborate description including standard sequential and direct photoionization channels. Both approaches are based on rate equations and take into account a Gaussian spatial intensity distribution of the laser beam. From the comparison we conclude that photoionization up to a charge of 5+ can be described by the minimal model which we interpret as sequential photoionization assisted by electron shake-up processes. For higher charges, the experimental ionization yields systematically exceed the elaborate rate-based prediction.

Intrinsic photosensitizer fluorescence measured using multi-diameter single-fiber spectroscopy in vivo

NASA Astrophysics Data System (ADS)

van Leeuwen-van Zaane, Floor; Gamm, Ute A.; van Driel, Pieter B. A. A.; Snoeks, Thomas J.; de Bruijn, Henriette S.; van der Ploeg-van den Heuvel, Angelique; Sterenborg, Henricus J. C. M.; Löwik, Clemens W.; Amelink, Arjen; Robinson, Dominic J.

2014-01-01

Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48] h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.

An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

2017-03-01

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in

An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy.

PubMed

Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

2017-01-18

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in

Fluorescence recognition of chiral amino alcohols by using a novel ionic liquid sensor.

PubMed

Cai, Pengfei; Wu, Datong; Zhao, Xiaoyong; Pan, Yuanjiang

2017-08-07

A novel task-specific ionic liquid derived from l-phenylalaninol was prepared as an enantioselective fluorescent sensor for the first time. Fluorescent chiral ionic liquid 1 (FCIL1) is found to exhibit highly enantioselective fluorescence enhancements toward both aromatic and non-aromatic chiral amino alcohols. When (S)-FCIL1 was treated with the enantiomers of phenylalaninol, a great fluorescence enhancement at 349 nm could be observed and the value of the enantiomeric fluorescence difference (ef) is 5.92. This demonstrated that the chiral sensor (S)-FCIL1 exhibited an excellent enantioselective response behaviour to d-phenylalaninol. Besides that, both the fluorescence intensity at 349 nm (I 349 ) and the ratio of I 349 to I 282 depend linearly on the concentration of amino alcohols. Both the concentration and the enantiomeric composition could be determined by using the chiral ionic liquid. Differently, the sensor treated with the enantiomers of 2-amino-1-butanol showed an opposite result: the fluorescence intensity of the S-enantiomer is higher than that of the R-enantiomer. Furthermore, the size of the substituents on the chiral carbon might be important for the enantioselective fluorescent response.

Photodynamic therapy and fluorescent diagnostics of skin cancer with radochlorine and photosense: comparing efficacy and toxicity

NASA Astrophysics Data System (ADS)

Vakulovskaya, Elena G.; Kemov, Yuriy V.; Zalevsky, Igor D.; Reshetnikov, Andrew V.; Umnova, Loubov V.; Vorozhcsov, Georgiu N.

2004-06-01

Photodynamic therapy (PDT) and fluorescent diagnostics (FD) with Radaclorine (RadaPharma, Russia) (RC) have been provided in 32 patients with T1-4 stage basal cell carcinoma (BCC) and in 81 patients with Photsense. Pharmacocynetic studies with detecting the borders of tumor growth and intensity of accumulation of photosensizers in tumor, normal tissues and visualization have been done by Spectral-fluorescent Complex and spectranalyser LESA-01 (He-Ne-laser, λ=633nm). We've got fluorescence of all tumors and additional fluorescence zones were found, cytological verification of BCC was got in most of cases. The fluorescent signs of RC in normal skin were found till 5 days after injection. As a source of light for PDT we used simeconductive lasers: Milon - λ = 660+2nm, light dose was 200-300 J/cm2 and Biospec (λ+672+2nm), multiple laser surface and interstitial irradiation was performed 24 hours after PS injection with total light dose till 400-600 J/cm2. 2 months after PDT with RC complete response (CR) in 65.6% of cases, partial response-in 34.4% of cases. The efficacy of PDT with PS was higher (CR-84.0%, PR-14.8%). Our experience show pronounced efficacy of PDT with RC for BCC without side effects and very short skin toxicity.

Experimental recovery of intrinsic fluorescence and fluorophore concentration in the presence of hemoglobin: spectral effect of scattering and absorption on fluorescence

NASA Astrophysics Data System (ADS)

Du Le, Vinh Nguyen; Patterson, Michael S.; Farrell, Thomas J.; Hayward, Joseph E.; Fang, Qiyin

2015-12-01

The ability to recover the intrinsic fluorescence of biological fluorophores is crucial to accurately identify the fluorophores and quantify their concentrations in the media. Although some studies have successfully retrieved the fluorescence spectral shape of known fluorophores, the techniques usually came with heavy computation costs and did not apply for strongly absorptive media, and the intrinsic fluorescence intensity and fluorophore concentration were not recovered. In this communication, an experimental approach was presented to recover intrinsic fluorescence and concentration of fluorescein in the presence of hemoglobin (Hb). The results indicated that the method was efficient in recovering the intrinsic fluorescence peak and fluorophore concentration with an error of 3% and 10%, respectively. The results also suggested that chromophores with irregular absorption spectra (e.g., Hb) have more profound effects on fluorescence spectral shape than chromophores with monotonic absorption and scattering spectra (e.g., black India ink and polystyrene microspheres).

Fluorescence evolution of leachates during treatment processes from two contrasting landfills.

PubMed

Sun, W L; Liu, T T; Cui, F; Ni, J R

2008-10-01

Landfill leachates are composed of a complex mixture of organic matter, including a wide range of potentially fluorescent organic compounds. The fluorescence excitation-emission matrix (FEEM) of leachates during treatment processes is investigated. Particular attention is paid to the fluorescence evolution of leachates during treatment processes. Two typical types of landfill, landfill A (a direct municipal solid waste (MSW) landfill) and landfill B (disposal of bottom ashes from MSW incinerators), in a city in Southern China were selected. The results show that two characteristic and intense excitation-emission peaks located at Ex/Em = 310-330 nm/395-410 nm (peak alpha) and Ex/Em = 250-260 nm/450-460 nm (peak alpha') are observed. As the aromatic chemicals, capable of emitting fluorescence, are more recalcitrant to biodegradation than aliphatic chemicals, enhancement of the dissolved organic carbon normalized fluorescence intensities is demonstrated during treatment processes of leachate A and leachate B. This is confirmed by the variation of ultraviolet absorptivity of leachates at 254 nm. Peak alpha' and peak alpha are attributed to a mixture of xenobiotic organic compounds with low molecular weight and relatively stable aromatic fulvic-like matters with high molecular weight, respectively. Humic substances are more resistant to biodegradation than xenobiotic organic compounds, so a significant reduction in the Ialpha'/Ialpha values (fluorescence intensity ratios of peak alpha' and peak a) of leachate A was observed during treatment processes. However, no evident variation for the Ialpha/Ialpha values of leachate B was found during treatment processes owing to the low concentrations of xenobiotic organic compounds in leachate B after incineration.

X-ray luminescence computed tomography imaging via multiple intensity weighted narrow beam irradiation

NASA Astrophysics Data System (ADS)

Feng, Bo; Gao, Feng; Zhao, Huijuan; Zhang, Limin; Li, Jiao; Zhou, Zhongxing

2018-02-01

The purpose of this work is to introduce and study a novel x-ray beam irradiation pattern for X-ray Luminescence Computed Tomography (XLCT), termed multiple intensity-weighted narrow-beam irradiation. The proposed XLCT imaging method is studied through simulations of x-ray and diffuse lights propagation. The emitted optical photons from X-ray excitable nanophosphors were collected by optical fiber bundles from the right-side surface of the phantom. The implementation of image reconstruction is based on the simulated measurements from 6 or 12 angular projections in terms of 3 or 5 x-ray beams scanning mode. The proposed XLCT imaging method is compared against the constant intensity weighted narrow-beam XLCT. From the reconstructed XLCT images, we found that the Dice similarity and quantitative ratio of targets have a certain degree of improvement. The results demonstrated that the proposed method can offer simultaneously high image quality and fast image acquisition.

Fluorescence enhancement of fluorescent unnatural streptavidin by binding of a biotin analogue with spacer tail and its application to biotin sensing.

PubMed

Zhu, Xianwei; Shinohara, Hiroaki

2014-01-01

We designed a novel molecular biosensing system for the detection of biotin, an important vitamin by the combination of fluorescent unnatural streptavidin with a commercialized biotin-(AC5)2-hydrazide. A fluorescent unnatural amino acid, BODIPY-FL-aminophenylalanine (BFLAF), was position-specifically incorporated into Trp120 of streptavidin by four-base codon method. Fluorescence of the Trp120BFLAF mutant streptavidin was enhanced by the addition of biotin-(AC5)2-hydrazide with the concentration dependent, whereas fluorescence enhancement was not observed at all by the addition of natural biotin. It was considered that the spacer tail of biotin-(AC5)2-hydrazide may disturb the fluorescence quenching of the Trp120BFLAF by Trp79 and Trp108 of the neighbor subunit. Therefore, biotin sensing was carried out by the competitive binding reaction of biotin-(AC5)2-hydrazide and natural biotin to the fluorescent mutant streptavidin. The fluorescence intensity decreased by increasing free biotin concentration. The result suggested that molecular biosensor for small ligand could be successfully designed by the pair of fluorescent mutant binding protein and ligand analogue.

Automatic cytometric device using multiple wavelength excitations

NASA Astrophysics Data System (ADS)

Rongeat, Nelly; Ledroit, Sylvain; Chauvet, Laurence; Cremien, Didier; Urankar, Alexandra; Couderc, Vincent; Nérin, Philippe

2011-05-01

Precise identification of eosinophils, basophils, and specific subpopulations of blood cells (B lymphocytes) in an unconventional automatic hematology analyzer is demonstrated. Our specific apparatus mixes two excitation radiations by means of an acousto-optics tunable filter to properly control fluorescence emission of phycoerythrin cyanin 5 (PC5) conjugated to antibodies (anti-CD20 or anti-CRTH2) and Thiazole Orange. This way our analyzer combining techniques of hematology analysis and flow cytometry based on multiple fluorescence detection, drastically improves the signal to noise ratio and decreases the spectral overlaps impact coming from multiple fluorescence emissions.

MEH-PPV film thickness influenced fluorescent quenching of tip-coated plastic optical fiber sensors

NASA Astrophysics Data System (ADS)

Yusufu, A. M.; Noor, A. S. M.; Tamchek, N.; Abidin, Z. Z.

2017-12-01

The performance of plastic optical fiber sensors in detecting nitro aromatic explosives 1,4-dinitrobenzene (DNB) have been investigated by fluorescence spectroscopy and analyzed by using fluorescence quenching technique. The plastic optical fiber utilized is 90 degrees cut tip and dip-coated with conjugated polymer MEH-PPV poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] thin films for detection conjugants. The thicknesses of the MEH-PPV coating were varied to improvise the sensitivity whilst slowly reducing the fluorescence intensity. It was shown that fluorescence intensity from thinner film decreased by (82% in 40 s) in the presence of DNB signifying an improvement of 28% reduction with time 13 s less than that of the thicker film.

Fluorescent IgG fusion proteins made in E. coli

PubMed Central

Luria, Yael; Raichlin, Dina; Benhar, Itai

2012-01-01

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals. PMID:22531449

Multispectral fluorescence image algorithms for detection of frass on mature tomatoes

USDA-ARS?s Scientific Manuscript database

A multispectral algorithm derived from hyperspectral line-scan fluorescence imaging under violet LED excitation was developed for the detection of frass contamination on mature tomatoes. The algorithm utilized the fluorescence intensities at five wavebands, 515 nm, 640 nm, 664 nm, 690 nm, and 724 nm...

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

PubMed Central

Lindhoud, Simon; Westphal, Adrie H.; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Borst, Jan Willem

2014-01-01

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

PubMed

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Shedding quantitative fluorescence light on novel regulatory mechanisms in skeletal biomedicine and biodentistry.

PubMed

Lee, Ji-Won; Iimura, Tadahiro

2017-02-01

Digitalized fluorescence images contain numerical information such as color (wavelength), fluorescence intensity and spatial position. However, quantitative analyses of acquired data and their validation remained to be established. Our research group has applied quantitative fluorescence imaging on tissue sections and uncovered novel findings in skeletal biomedicine and biodentistry. This review paper includes a brief background of quantitative fluorescence imaging and discusses practical applications by introducing our previous research. Finally, the future perspectives of quantitative fluorescence imaging are discussed.

Photodynamic therapy and fluorescent diagnostics of head and neck cancer with second-generation photosensitizers

NASA Astrophysics Data System (ADS)

Vakulovskaya, Elena G.

2005-08-01

Photodynamic Therapy (PDT) and fluorescent diagnostics (FD) using Photosense was provided in 50 patients with head and neck cancer T1-3 stage, in 89 patients with skin cancer, using Radaclorine (RC) in 42 patients with T1-4 stage basal cell carcinoma (BCC),in 6 patients with oral cancer. Detection of borders of tumor, intensity of accumulation of photosensitizers in tumor, normal tissues were done by Spectral-fluorescent Complex. We"ve got fluorescence o fa 11 tumors and additional fluorescence zones were found with cytological verification. We used semiconductive lasers: Milon - h = 660+2nm, light dose 200 - 300 J/cm2 and Biospec (h=672+2nm), multiple laser surface and interstitial irradiation with total 1 ight d ose till 4 00-600 Ji cm2. A fter P DT with P S in head and neck cancer we"ve had complete response (CR) in 66.0% and partial response (PR) in 30.0%, with RC CR in BCC T1- 2NOMO - 92.9%, in recurrrencies CR - 60,6%, PR - 39,4%. The efficacy of PDT with PS was higher (CR - 86.7%, PR - 13,3%) and the recurrence rate in 6 months lower. Our experience show pronounced efficacy of PDT for head and neck tumors of different localization and histology, FD is providing diagnostically significant information, demonstrated high sensitivity and specificity.

A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe.

PubMed

Liu, Hua; Na, Weidan; Liu, Ziping; Chen, Xueqian; Su, Xingguang

2017-06-15

In this paper, a facile and rapid fluorescence turn-on assay for fluorescent detection of ascorbic acid (AA) was developed by using the orange emission graphene quantum dots (GQDs). In the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ), catechol can be oxidized by hydroxyl radicals and converted to o-benzoquinone, which can significantly quench the fluorescence of GQDs. However, when AA present in the system, it can consume part of H 2 O 2 and hydroxyl radicals to inhibit the generation of o-benzoquinone, resulting in fluorescence recovery. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of H 2 O 2 in the range of 3.33-500µM with a detection limit of 1.2µM. The linear detection for AA was in the range from 1.11 to 300µM with a detection limit of 0.32µM. The proposed method was applied to the determination of AA in human serum samples with satisfactory results. Copyright © 2017. Published by Elsevier B.V.

Fluorescence optical imaging in anticancer drug delivery.

PubMed

Etrych, Tomáš; Lucas, Henrike; Janoušková, Olga; Chytil, Petr; Mueller, Thomas; Mäder, Karsten

2016-03-28

In the past several decades, nanosized drug delivery systems with various targeting functions and controlled drug release capabilities inside targeted tissues or cells have been intensively studied. Understanding their pharmacokinetic properties is crucial for the successful transition of this research into clinical practice. Among others, fluorescence imaging has become one of the most commonly used imaging tools in pre-clinical research. The development of increasing numbers of suitable fluorescent dyes excitable in the visible to near-infrared wavelengths of the spectrum has significantly expanded the applicability of fluorescence imaging. This paper focuses on the potential applications and limitations of non-invasive imaging techniques in the field of drug delivery, especially in anticancer therapy. Fluorescent imaging at both the cellular and systemic levels is discussed in detail. Additionally, we explore the possibility for simultaneous treatment and imaging using theranostics and combinations of different imaging techniques, e.g., fluorescence imaging with computed tomography. Copyright © 2016 Elsevier B.V. All rights reserved.

Holographic fluorescence mapping using space-division matching method

NASA Astrophysics Data System (ADS)

Abe, Ryosuke; Hayasaki, Yoshio

2017-10-01

Three-dimensional mapping of fluorescence light sources was performed by using self-interference digital holography. The positions of the sources were quantitatively determined by using Gaussian fitting of the axial and lateral intensity distributions obtained from diffraction calculations through position calibration from the observation space to the sample space. A space-division matching method was developed to perform the mapping of many fluorescence light sources, in this experiment, 500 nm fluorescent nanoparticles fixed in gelatin. A fluorescence digital holographic microscope having a 60 × objective lens with a numerical aperture of 1.25 detected 13 fluorescence light sources in a measurable region with a radius of ∼ 20 μm and a height of ∼ 5 μm. It was found that the measurable region had a conical shape resulting from the overlap between two beams.

Dual-detector X-ray fluorescence imaging of ancient artifacts with surface relief

PubMed Central

Smilgies, Detlef-M.; Powers, Judson A.; Bilderback, Donald H.; Thorne, Robert E.

2012-01-01

Interpretation of X-ray fluorescence images of archeological artifacts is complicated by the presence of surface relief and roughness. Using two symmetrically arranged fluorescence detectors in a back-reflection geometry, the proper X-ray fluorescence yield can be distinguished from intensity variations caused by surface topography. This technique has been applied to the study of Roman inscriptions on marble. PMID:22713888

Development of a fluorescent chelating ligand for scandium ion having a Schiff base moiety

NASA Astrophysics Data System (ADS)

Yamada, Hiroshi; Kojo, Masahito; Nakahara, Tomomi; Murakami, Kumi; Kakima, Takashi; Ichiba, Hideaki; Yajima, Takehiko; Fukushima, Takeshi

2012-05-01

A fluorescent ligand, 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone (HMB-ASH), was newly designed and synthesized, and its fluorescence characteristics for metal ions were investigated in the pH range 3.0-10.5 (at a difference of 0.5 for each metal). After testing 31 different metal ions, it was found that HMB-ASH was able to emit fluorescence intensely at 512 nm with an excitation wavelength of 353 nm in the presence of Sc3+, one of the rare earth metals, at pH values around 3.5 and 8.0. The other metal ions hardly showed fluorescence with HMB-ASH. The fluorescence was more intense at pH 8.0, and the detection limit of Sc3+ in a buffer solution (pH 8.0) was approximately 18.8 nmol L-1 (0.85 ppb).

Study on excitation and fluorescence spectrums of Japanese citruses to construct machine vision systems for acquiring fluorescent images

NASA Astrophysics Data System (ADS)

Momin, Md. Abdul; Kondo, Naoshi; Kuramoto, Makoto; Ogawa, Yuichi; Shigi, Tomoo

2011-06-01

Research was conducted to acquire knowledge of the ultraviolet and visible spectrums from 300 -800 nm of some common varieties of Japanese citrus, to investigate the best wave-lengths for fluorescence excitation and the resulting fluorescence wave-lengths and to provide a scientific background for the best quality fluorescent imaging technique for detecting surface defects of citrus. A Hitachi U-4000 PC-based microprocessor controlled spectrophotometer was used to measure the absorption spectrum and a Hitachi F-4500 spectrophotometer was used for the fluorescence and excitation spectrums. We analyzed the spectrums and the selected varieties of citrus were categorized into four groups of known fluorescence level, namely strong, medium, weak and no fluorescence.The level of fluorescence of each variety was also examined by using machine vision system. We found that around 340-380 nm LEDs or UV lamps are appropriate as lighting devices for acquiring the best quality fluorescent image of the citrus varieties to examine their fluorescence intensity. Therefore an image acquisition device was constructed with three different lighting panels with UV LED at peak 365 nm, Blacklight blue lamps (BLB) peak at 350 nm and UV-B lamps at peak 306 nm. The results from fluorescent images also revealed that the findings of the measured spectrums worked properly and can be used for practical applications such as for detecting rotten, injured or damaged parts of a wide variety of citrus.

The fluorescence properties of aerosol larger than 0.8 μm in urban and tropical rainforest locations

NASA Astrophysics Data System (ADS)

Gabey, A. M.; Stanley, W. R.; Gallagher, M. W.; Kaye, P. H.

2011-06-01

UV-LIF measurements were performed on ambient aerosol in Manchester, UK (urban city centre, winter) and Borneo, Malaysia (remote, tropical) using a Wide Issue Bioaerosol Spectrometer, version 3 (WIBS3). These sites are taken to represent environments with minor and significant primary biological aerosol (PBA) influences respectively, and the urban dataset describes the fluorescent background aerosol against which PBA must be identified by researchers using LIF. The ensemble aerosol at both sites was characterised over 2-3 weeks by measuring the fluorescence intensity and optical equivalent diameter (DP) of single particles sized 0.8 ≤ DP ≤ 20 μm. Filter samples were also collected for a subset of the Manchester campaign and analysed using energy dispersive X-Ray (EDX) spectroscopy and environmental scanning electron microscopy (ESEM), which revealed mostly non-PBA at D ≤ 1 μm. The WIBS3 features three fluorescence channels: the emission following a 280 nm excitation is recorded at 310-400 nm (channel F1) and 400-600 nm (F2), and fluorescence excited at 350 nm is detected at 400-600 nm (F3). In Manchester the primary size mode of fluorescent and non-fluorescent material was present at 0.8-1.2 μm, with a secondary fluorescent mode at 2-4 μm. In Borneo non-fluorescent material peaked at 0.8-1.2 μm and fluorescent at 3-4 μm. Agreement between fluorescent number concentrations in each channel differed at the two sites, with F1 and F3 reporting similar concentrations in Borneo but F3 outnumbering F1 by a factor of 2-3 across the size spectrum in Manchester. The fluorescence intensity in each channel generally rose with DP at both sites with the exception of F1 intensity in Manchester, which peaked at DP = 4 μm, causing a divergence between F1 and F3 intensity at larger DP. This divergence and the differing fluorescent particle concentrations demonstrate the additional discrimination provided by the F1 channel in Manchester. The relationships between

Characterization of Fluorescent Polystyrene Microspheres for Advanced Flow Diagnostics

NASA Technical Reports Server (NTRS)

Maisto, Pietro M. F.; Lowe, K. Todd; Byun, Guibo; Simpson, Roger; Vercamp, Max; Danley, Jason E.; Koh, Brian; Tiemsin, Pacita; Danehy, Paul M.; Wohl, Christopher J.

2013-01-01

Fluorescent dye-doped polystyrene latex microspheres (PSLs) are being developed for velocimetry and scalar measurements in variable property flows. Two organic dyes, Rhodamine B (RhB) and dichlorofluorescence (DCF), are examined to assess laser-induced fluorescence (LIF) properties for flow imaging applications and single-shot temperature measurements. A major interest in the current research is the application of safe dyes, thus DCF is of particular interest, while RhB is used as a benchmark. Success is demonstrated for single-point laser Doppler velocimetry (LDV) and also imaging fluorescence, excited via a continuous wave 2 W laser beam, for exposures down to 10 ms. In contrast, when exciting with a pulsed Nd:YAG laser at 200 mJ/pulse, no fluorescence was detected, even when integrating tens of pulses. We show that this is due to saturation of the LIF signal at relatively low excitation intensities, 4-5 orders of magnitude lower than the pulsed laser intensity. A two-band LIF technique is applied in a heated jet, indicating that the technique effectively removes interfering inputs such as particle diameter variation. Temperature measurement uncertainties are estimated based upon the variance measured for the two-band LIF intensity ratio and the achievable dye temperature sensitivity, indicating that particles developed to date may provide about +/-12.5 C precision, while future improvements in dye temperature sensitivity and signal quality may enable single-shot temperature measurements with sub-degree precision.

Identification of Atherosclerotic Plaques in Carotid Artery by Fluorescence Spectroscopy

NASA Astrophysics Data System (ADS)

Rocha, Rick; Villaverde, Antonio Balbin; Silveira, Landulfo; Costa, Maricília Silva; Alves, Leandro Procópio; Pasqualucci, Carlos Augusto; Brugnera, Aldo

2008-04-01

The aim of this work was to identify the presence of atherosclerotic plaques in carotid artery using the Fluorescence Spectroscopy. The most important pathogeny in the cardiovascular disorders is the atherosclerosis, which may affect even younger individuals. With approximately 1.2 million heart attacks and 750,000 strokes afflicting an aging American population each year, cardiovascular disease remains the number one cause of death. Carotid artery samples were obtained from the Autopsy Service at the University of São Paulo (São Paulo, SP, Brazil) taken from cadavers. After a histopathological analysis the 60 carotid artery samples were divided into two groups: normal (26) and atherosclerotic plaques (34). Samples were irradiated with the wavelength of 488 nm from an Argon laser. A 600 μm core optical fiber, coupled to the Argon laser, was used for excitation of the sample, whereas another 600 optical fiber, coupled to the spectrograph entrance slit, was used for collecting the fluorescence from the sample. Measurements were taken at different points on each sample and then averaged. Fluorescence spectra showed a single broad line centered at 549 nm. The fluorescence intensity for each sample was calculated by subtracting the intensity at the peak (550 nm) and at the bottom (510 nm) and then data were statistically analyzed, looking for differences between both groups of samples. ANOVA statistical test showed a significant difference (p<0,05) between both types of tissues, with regard to the fluorescence peak intensities. Our results indicate that this technique could be used to detect the presence of the atherosclerotic in carotid tissue.

A theoretical investigation of single-molecule fluorescence detection on thin metallic layers.

PubMed

Enderlein, J

2000-04-01

In the present paper, the excitation and detection of single-molecule fluorescence over thin metallic films is studied theoretically within the framework of classical electrodynamics. The model takes into account the specific conditions of surface plasmon-assisted optical excitation, fluorescence quenching by the metal film, and detection geometry. Extensive numerical results are presented for gold, silver, and aluminum films, showing the detectable fluorescence intensities and their dependence on film thickness and the fluorescent molecule's position under optimal excitation conditions.

An investigation of nonsimultaneous laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Fletcher, D. G.

1993-01-01

An alternative to simultaneous, two-line laser-induced fluorescence for thermodynamic property measurement is presented. This spectroscopic approach is similar to multiple-overheat hot-wire anemometry and is based on laser excitation of different fluorescence transitions for separate, sequential wind tunnel runs. Both fluctuating and mean thermodynamic property measurements seem to be achievable with this method without exciting the transitions during the same laser pulse.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, Edward S.; Taylor, John A.

1996-03-12

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, Edward S.; Taylor, John A.

1994-06-28

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

Development of a fluorescent x-ray source for medical imaging

NASA Astrophysics Data System (ADS)

Toyofuku, F.; Tokumori, K.; Nishimura, K.; Saito, T.; Takeda, T.; Itai, Y.; Hyodo, K.; Ando, M.; Endo, M.; Naito, H.; Uyama, C.

1995-02-01

A fluorescent x-ray source for medical imaging, such as K-edge subtraction angiography and monochromatic x-ray CT, has been developed. Using a 6.5 GeV accumulation ring in Tsukuba, fluorescent x rays, which range from about 30 to 70 keV are generated by irradiating several target materials. Measurements have been made of output intensities and energy spectra for different target angles and extraction angles. The intensities of fluorescent x rays at a 30 mA beam current are on the order of 1-3×106 photons/mm2/s at 30 cm from the local spot where the incident beam is collimated to 1 mm2. A phantom which contains three different contrast media (iodine, barium, gadolinium) was used for the K-edge energy subtraction, and element selective CT images were obtained.

[Simultaneous determination of multiple elements in airborne particulate samples by X-ray fluorescence spectrometry].

PubMed

Takada, T; Hitosugi, M; Kadowaki, T; Kudo, M

1983-07-01

An energy dispersive X-ray fluorescence spectrometer (EDX) has been applied to determine multielements in the workplace air. The standards for X-ray fluorescence analysis were prepared by the chelate precipitation method on polyvinyl chloride (PVC) membrane filter. And, the specimens were prepared to deposit various metal compounds of different chemical forms by the suspension method on PVC membrane filter, and they were determined with EDX and atomic absorption spectrometer (AAS). The results obtained were as follows. Though there is a difference by each element, an amount less than 3 microgram/cm2 per unit area makes it possible to undergo multielement analysis, that is, is has no influence on fine particle effect (particle size; under 5 microns). Then, effects of the X-ray intensity by different chemical forms are negligible. At the presence the neighboring element and other elements this technique showed greater precision by carrying out on corrective treatment, etc. The coefficient of variation of this technique was in the range of 2.5-6.5% at DDTC-Cu of 0.5-5.0 micrograms/cm2, with the limit of detection for As : 0.002 microgram/cm2, Zn : 0.003 microgram/cm2, Pb : 0.003 microgram/cm2, Cu : 0.004 microgram/cm2, Ni : 0.003 microgram/cm2, Fe : 0.005 microgram/cm2, Mn : 0.008 microgram/cm2, Cr : 0.013 microgram/cm2, respectively. Aerosols collected at the workplace were analyzed with EDX and AAS, and the obtained results showed good agreement with such regression line as y = 1.04 chi + 0.04, the coefficient of correlation being r = 0.995. From these results, this technique was found to be a very excellent method for monitoring of multielements in the workplace air.

ALA-induced fluorescence in the canine oral cavity.

PubMed

Vaidyanathan, Vijay; Wiggs, Robert; Stohl, Josh; Baxi, Mehul

2006-06-01

We examined whether 5-aminolevulinic acid (ALA) could enhance the spectroscopic contrast between normal and diseased oral tissues, without prolonged photosensitivity. ALA is a promising photosensitizing agent. Adose of 25 mg/kg of ALA was administered intravenously to five dogs with gingivitis and three dogs with oral cancer, respectively. Fluorescence was recorded from the diseased sites in the oral cavity in addition to normal sites. ALA-induced proto-porphyrin IX fluorescence at all gingivitis sites reached a peak in 2-3 h and returned to baseline in 24 h. Fluorescence from the gingivitis site was observed earlier and was higher than the fluorescence from the normal site. For dogs with cancer, fluorescence from the cancerous sites occurred earlier in time compared to gingivitis sites and was comparatively higher in intensity. The fluorescence from the diseased sites was found to be higher than the normal site. Clinical and fluorescence data suggest that a dose of 25 mg/kg may be satisfactory for diagnostic purposes and would have minimal side effects.

Studying Photosynthesis by Measuring Fluorescence

ERIC Educational Resources Information Center

Sanchez, Jose Francisco; Quiles, Maria Jose

2006-01-01

This paper describes an easy experiment to study the absorption and action spectrum of photosynthesis, as well as the inhibition by heat, high light intensity and the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the photosynthetic process. The method involves measuring the chlorophyll fluorescence emitted by intact…

Genetic Analysis of Digestive Physiology Using Fluorescent Phospholipid Reporters

NASA Astrophysics Data System (ADS)

Farber, Steven A.; Pack, Michael; Ho, Shiu-Ying; Johnson, Iain D.; Wagner, Daniel S.; Dosch, Roland; Mullins, Mary C.; Hendrickson, H. Stewart; Hendrickson, Elizabeth K.; Halpern, Marnie E.

2001-05-01

Zebrafish are a valuable model for mammalian lipid metabolism; larvae process lipids similarly through the intestine and hepatobiliary system and respond to drugs that block cholesterol synthesis in humans. After ingestion of fluorescently quenched phospholipids, endogenous lipase activity and rapid transport of cleavage products results in intense gall bladder fluorescence. Genetic screening identifies zebrafish mutants, such as fat free, that show normal digestive organ morphology but severely reduced phospholipid and cholesterol processing. Thus, fluorescent lipids provide a sensitive readout of lipid metabolism and are a powerful tool for identifying genes that mediate vertebrate digestive physiology.

Specific detection of Vibrio parahaemolyticus by fluorescence quenching immunoassay based on quantum dots.

PubMed

Wang, Ling; Zhang, Junxian; Bai, Haili; Li, Xuan; Lv, Pintian; Guo, Ailing

2014-07-01

In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43%. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC · HCl, 150 μL of 4 mg/mL NHS, buffer system of Na2HPO4-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764%. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity.

Copper nanoclusters as probes for turn-on fluorescence sensing of L-lysine.

PubMed

Zhang, Mingming; Qiao, Juan; Zhang, Shufeng; Qi, Li

2018-05-15

Herein, a unique protocol based on copper nanoclusters (CuNCs) probe for turn-on fluorescence sensing of L-lysine was developed. The fluorescent CuNCs with ovalbumin as the stabilizer was prepared by a simple, one-step and green method. When 370 nm was used as the excitation wavelength, the resultant CuNCs exhibited a pale blue fluorescence with the maximum emission at 440 nm. Interestingly, existence of L-lysine evoked the obvious fluorescence intensity increase of CuNCs. The detection limit of the proposed method for L-lysine was 5.5 μM, with a good linear range from 10.0 μM to 1.0 mM (r 2 = 0.999). Moreover, the possible mechanism for enhanced fluorescence intensity of CuNCs by addition of L-lysine was explored and discussed briefly. Further, the as-prepared fluorescent CuNCs was successfully applied in detection of L-lysine in urine. Our results demonstrated that L-lysine could be monitored by the probe, providing new path for construction of CuNCs as fluorescent probes and showing great potential in quantification of L-lysine in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Green Tea Catechins Quench the Fluorescence of Bacteria-Conjugated Alexa Fluor Dyes

PubMed Central

Zhao, Lin; Li, Wei; Zhu, Shu; Tsai, Sheena; Li, Jianhua; Tracey, Kevin J.; Wang, Ping; Fan, Saijun; Sama, Andrew E.; Wang, Haichao

2013-01-01

Accumulating evidence suggests that Green tea polyphenolic catechins, especially the (-)-epigallocatechin gallate (EGCG), can be cross-linked to many proteins, and confer a wide range of anti-bacterial activities possibly by damaging microbial cytoplasmic lipids and proteins. At the doses that conferred protection against lethal polymicrobial infection (induced by cecal ligation and puncture), EGCG significantly reduced bacterial loads particularly in the liver and lung. To elucidate its bactericidal mechanisms, we determined whether EGCG affected the fluorescence intensities of bacteria-conjugated Alexa Fluor 488 or 594 dyes. When mixed with unconjugated Alexa Fluor 488 or 594 dyes, EGCG or analogs did not affect the fluorescence intensity of these dyes. In a sharp contrast, EGCG and some analogs (e.g., Catechin Gallate, CG), markedly reduced the fluorescence intensity of Gram-positive Staphylococcus aureus-conjugated Alexa 594 and Gram-negative Escherichia coli-conjugated Alexa 488. Interestingly, co-treatment with ethanol impaired the EGCG-mediated fluorescence quenching of the G+ S. aureus, but not of the G- E. coli-conjugated Alexa Flour dyes. In light of the notion that Alexa Fluor dyes can be quenched by aromatic amino acids, it is plausible that EGCG exerts anti-microbial activities possibly by altering microbial protein conformations and functions. This possibility can now be explored by screening other fluorescence-quenching agents for possible antimicrobial activities. PMID:24011199

Application of normal fluorescence and stability-indicating derivative synchronous fluorescence spectroscopy for the determination of gliquidone in presence of its fluorescent alkaline degradation product

NASA Astrophysics Data System (ADS)

El-ghobashy, Mohamed R.; Yehia, Ali M.; Helmy, Aya H.; Youssef, Nadia F.

2018-01-01

Simple, smart and sensitive normal fluorescence and stability-indicating derivative synchronous spectrofluorimetric methods have been developed and validated for the determination of gliquidone in the drug substance and drug product. Normal spectrofluorimetric method of gliquidone was established in methanol at λ excitation 225 nm and λ emission 400 nm in concentration range 0.2-3 μg/ml with LOD equal 0.028. The fluorescence quantum yield of gliquidone was calculated using quinine sulfate as a reference and found to be 0.542. Stability-indicating first and third derivative synchronous fluorescence spectroscopy were successfully utilized to overcome the overlapped spectra in normal fluorescence of gliquidone and its alkaline degradation product. Derivative synchronous methods are based on using the synchronous fluorescence of gliquidone and its degradation product in methanol at Δ λ50 nm. Peak amplitude in the first derivative of synchronous fluorescence spectra was measured at 309 nm where degradation product showed zero-crossing without interference. The peak amplitudes in the third derivative of synchronous fluorescence spectra, peak to trough were measured at 316,329 nm where degradation product showed zero-crossing. The different experimental parameters affecting the normal and synchronous fluorescence intensity of gliquidone were studied and optimized. Moreover, the cited methods have been validated as per ICH guidelines. The peak amplitude-concentration plots of the derivative synchronous fluorescence were linear over the concentration range 0.05-2 μg/ml for gliquidone. Limits of detection were 0.020 and 0.022 in first and third derivative synchronous spectra, respectively. The adopted methods were successfully applied to commercial tablets and the results demonstrated that the derivative synchronous fluorescence spectroscopy is a powerful stability-indicating method, suitable for routine use with a short analysis time. Statistical comparison between

Optimization via specific fluorescence brightness of a receptor-targeted probe for optical imaging and positron emission tomography of sentinel lymph nodes

PubMed Central

Qin, Zhengtao; Hall, David J.; Liss, Michael A.; Hoh, Carl K.; Kane, Christopher J.; Wallace, Anne M.

2013-01-01

Abstract. The optical properties of a receptor-targeted probe designed for dual-modality mapping of the sentinel lymph node (SLN) was optimized. Specific fluorescence brightness was used as the design criterion, which was defined as the fluorescence brightness per mole of the contrast agent. Adjusting the molar ratio of the coupling reactants, IRDye 800CW-NHS-ester and tilmanocept, enabled us to control the number of fluorescent molecules attached to each tilmanocept, which was quantified by H1 nuclear magnetic resonance spectroscopy. Quantum yields and molar absorptivities were measured for unconjugated IRDye 800CW and IRDye 800CW-tilmanocept (800CW-tilmanocept) preparations at 0.7, 1.5, 2.3, 2.9, and 3.8 dyes per tilmanocept. Specific fluorescence brightness was calculated by multiplication of the quantum yield by the molar absorptivity and the number of dyes per tilmanocept. It predicted that the preparation with 2.3 dyes per tilmanocept would exhibit the brightest signal, which was confirmed by fluorescence intensity measurements using three optical imaging systems. When radiolabeled with Ga68 and injected into the footpads of mice, the probe identified SLNs by both fluorescence and positron emission tomography (PET) while maintaining high percent extraction by the SLN. These studies demonstrated the feasibility of 800CW-tilmanocept for multimodal SLN mapping via fluorescence and PET–computed tomography imaging. PMID:23958947

Dynamic measurement of fluorescent proteins spectral distribution on virus infected cells

NASA Astrophysics Data System (ADS)

Lee, Ja-Yun; Wu, Ming-Xiu; Kao, Chia-Yun; Wu, Tzong-Yuan; Hsu, I.-Jen

2006-09-01

We constructed a dynamic spectroscopy system that can simultaneously measure the intensity and spectral distributions of samples with multi-fluorophores in a single scan. The system was used to monitor the fluorescence distribution of cells infected by the virus, which is constructed by a recombinant baculoviruses, vAcD-Rhir-E, containing the red and green fluorescent protein gene that can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. The system was composed of an excitation light source, a scanning system and a spectrometer. We also developed an algorithm and fitting process to analyze the pattern of fluorescence distribution of the dual fluorescence produced in the recombinant virus-infected cells. All the algorithm and calculation are automatically processed in a visualized scanning program and can monitor the specific region of sample by calculating its intensity distribution. The spectral measurement of each pixel was performed at millisecond range and the two dimensional distribution of full spectrum was recorded within several seconds. We have constructed a dynamic spectroscopy system to monitor the process of virus-infection of cells. The distributions of the dual fluorescence were simultaneously measured at micrometer resolution.

A Ratiometric Threshold for Determining Presence of Cancer During Fluorescence-guided Surgery

PubMed Central

Warram, Jason M; de Boer, Esther; Moore, Lindsay S.; Schmalbach, Cecelia E; Withrow, Kirk P; Carroll, William R; Richman, Joshua S; Morlandt, Anthony B; Brandwein-Gensler, Margaret; Rosenthal, Eben L

2015-01-01

Background&Objective Fluorescence-guided imaging to assist in identification of malignant margins has the potential to dramatically improve oncologic surgery. However a standardized method for quantitative assessment of disease-specific fluorescence has not been investigated. Introduced here is a ratiometric threshold derived from mean fluorescent tissue intensity that can be used to semi-quantitatively delineate tumor from normal tissue. Methods Open-field and a closed-field imaging devices were used to quantify fluorescence in punch biopsy tissues sampled from primary tumors collected during a phase 1 trial evaluating the safety of cetuximab-IRDye800 in patients (n=11) undergoing surgical intervention for head and neck cancer. Fluorescence ratios were calculated using mean fluorescence intensity (MFI) from punch biopsy normalized by MFI of patient-matched tissues. Ratios were compared to pathological assessment and a ratiometric threshold was established to predict presence of cancer. Results During open-field imaging using an intraoperative device, the threshold for muscle normalized tumor fluorescence was found to be 2.7, which produced a sensitivity of 90.5% and specificity of 78.6% for delineating disease tissue. The skin-normalized threshold generated greater sensitivity (92.9%) and specificity (81.0%). Conclusion Successful implementation of a semi-quantitative threshold can provide a scientific methodology for delineating disease from normal tissue during fluorescence-guided resection of cancer. PMID:26074273

A ratiometric threshold for determining presence of cancer during fluorescence-guided surgery.

PubMed

Warram, Jason M; de Boer, Esther; Moore, Lindsay S; Schmalbach, Cecelia E; Withrow, Kirk P; Carroll, William R; Richman, Joshua S; Morlandt, Anthony B; Brandwein-Gensler, Margaret; Rosenthal, Eben L

2015-07-01

Fluorescence-guided imaging to assist in identification of malignant margins has the potential to dramatically improve oncologic surgery. However, a standardized method for quantitative assessment of disease-specific fluorescence has not been investigated. Introduced here is a ratiometric threshold derived from mean fluorescent tissue intensity that can be used to semi-quantitatively delineate tumor from normal tissue. Open-field and a closed-field imaging devices were used to quantify fluorescence in punch biopsy tissues sampled from primary tumors collected during a phase 1 trial evaluating the safety of cetuximab-IRDye800 in patients (n = 11) undergoing surgical intervention for head and neck cancer. Fluorescence ratios were calculated using mean fluorescence intensity (MFI) from punch biopsy normalized by MFI of patient-matched tissues. Ratios were compared to pathological assessment and a ratiometric threshold was established to predict presence of cancer. During open-field imaging using an intraoperative device, the threshold for muscle normalized tumor fluorescence was found to be 2.7, which produced a sensitivity of 90.5% and specificity of 78.6% for delineating disease tissue. The skin-normalized threshold generated greater sensitivity (92.9%) and specificity (81.0%). Successful implementation of a semi-quantitative threshold can provide a scientific methodology for delineating disease from normal tissue during fluorescence-guided resection of cancer. © 2015 Wiley Periodicals, Inc.

Fluorescence chemodosimeter for dopamine based on the inner filter effect of the in situ generation of silver nanoparticles and fluorescent dye

NASA Astrophysics Data System (ADS)

Uppa, Yuwapon; Ngamdee, Kessarin; Promarak, Vinich; Ngeontae, Wittaya

2018-07-01

A new strategy for the sensitive and selective detection of dopamine (DA) was proposed. The chemodosimeter design was based on the measurement of the fluorescent quenching of fluorescein dye caused by the in situ generation of silver nanoparticles (AgNPs). The AgNPs can be simply generated by a reaction between DA and Ag+ in the presence of polymethacrylic acid (PMAA). In addition, the generated AgNPs possess the maximum surface plasmon resonance (SPR) at 440 nm and an increase in the SPR intensity with an increasing DA concentration. Basically, fluorescein dye can emit the fluorescent intensity maximum at 513 nm with excitation at 487 nm. Thus, fluorescent quenching was achieved due to an inner filter effect from the overlap between the excitation spectrum of the fluorescein dye and the SPR spectrum of the generated AgNPs. The degree of fluorescent quenching linearly depends on the number of generated AgNPs that can be directly related to the concentration of DA. The proposed chemodosimeter can be used to detect DA in a working linear concentration range of 1.0-5.0 μM at a detection limit of 10.6 nM. This chemodosimeter was successfully applied to determine DA in a real urine sample and a dopamine injection formulation with satisfactory results.