Real-time quantitative fluorescence imaging using a single snapshot optical properties technique for neurosurgical guidance

NASA Astrophysics Data System (ADS)

Valdes, Pablo A.; Angelo, Joseph; Gioux, Sylvain

2015-03-01

Fluorescence imaging has shown promise as an adjunct to improve the extent of resection in neurosurgery and oncologic surgery. Nevertheless, current fluorescence imaging techniques do not account for the heterogeneous attenuation effects of tissue optical properties. In this work, we present a novel imaging system that performs real time quantitative fluorescence imaging using Single Snapshot Optical Properties (SSOP) imaging. We developed the technique and performed initial phantom studies to validate the quantitative capabilities of the system for intraoperative feasibility. Overall, this work introduces a novel real-time quantitative fluorescence imaging method capable of being used intraoperatively for neurosurgical guidance.

Time-resolved fluorescence decay measurements for flowing particles

DOEpatents

Deka, C.; Steinkamp, J.A.

1999-06-01

Time-resolved fluorescence decay measurements are disclosed for flowing particles. An apparatus and method for the measurement and analysis of fluorescence for individual cells and particles in flow are described, wherein the rapid measurement capabilities of flow cytometry and the robust measurement and analysis procedures of time-domain fluorescence lifetime spectroscopy are combined. A pulse-modulated CW laser is employed for excitation of the particles. The characteristics and the repetition rate of the excitation pulses can be readily adjusted to accommodate for fluorescence decays having a wide range of lifetimes. 12 figs.

Time-resolved fluorescence decay measurements for flowing particles

DOEpatents

Deka, Chiranjit; Steinkamp, John A.

1999-01-01

Time-resolved fluorescence decay measurements for flowing particles. An apparatus and method for the measurement and analysis of fluorescence for individual cells and particles in flow are described, wherein the rapid measurement capabilities of flow cytometry and the robust measurement and analysis procedures of time-domain fluorescence lifetime spectroscopy are combined. A pulse-modulated cw laser is employed for excitation of the particles. The characteristics and the repetition rate of the excitation pulses can be readily adjusted to accommodate for fluorescence decays having a wide range of lifetimes.

Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors.

PubMed

Elsutohy, Mohamed M; Chauhan, Veeren M; Markus, Robert; Kyyaly, Mohammed Aref; Tendler, Saul J B; Aylott, Jonathan W

2017-05-11

Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

PubMed Central

Sapoznik, Etai; Niu, Guoguang; Zhou, Yu; Prim, Peter M.; Criswell, Tracy L.

2018-01-01

Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening. PMID:29444187

Real-time water and wastewater quality monitoring using LED-based fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Bridgeman, John; Zakharova, Yulia

2016-04-01

In recent years there have been a number of attempts to design and introduce into water management tools that are capable of measuring organic and microbial matter in real time and in situ. This is important, as the delivery of safe water to customers, and the discharge of good quality effluent to rivers are primary concerns to water undertakers. A novel, LED-based portable fluorimeter 'Duo Fluor' has been designed and constructed at the University of Birmingham to monitor the quality of (waste)water continuously and in real time, and its performance has been assessed in a range of environments. To be of use across a range of environments, special attention must be paid to two crucially important characteristics of such instruments, i.e. their sensitivity and robustness. Thus, the objectives of this study were: 1. To compare the performance (in terms of their sensitivity and robustness) of the Duo Fluor and two other commercial fluorescence devices in laboratory conditions. 2. To assess the performance of the Duo Fluor in situ, in real time at a 450,000PE WwTW. Initially, the impact of quinine sulphate (QS), a highly fluorescent alkaloid with high quantum fluorescence yield, on peak T fluorescence in environmental waters was examined for the Duo Fluor and two commercially available, chamber-based fluorimeters, (F1) and (F2). The instruments' responses to three scenarios were assessed: 1. Deionised water (DW) spiked with QS (from 0.05 to 0.4 mg/L); 2. Environmental water (pond water, PW) spiked with QS (from 0.05 to 0.4 mg/L); 3. Different water samples from various environmental source. The results show that the facility to amend gain settings and the suitable choice of gain are crucial to obtaining reliable data on both peaks T and C in a wide range of water types. The Duo Fluor offers both of these advantages whilst commercially available instruments currently do not. The Duo Fluor was subsequently fixed at the final effluent (FE) discharge point of a WwTW and FE

Comparative evaluation of performance measures for shading correction in time-lapse fluorescence microscopy.

PubMed

Liu, L; Kan, A; Leckie, C; Hodgkin, P D

2017-04-01

Time-lapse fluorescence microscopy is a valuable technology in cell biology, but it suffers from the inherent problem of intensity inhomogeneity due to uneven illumination or camera nonlinearity, known as shading artefacts. This will lead to inaccurate estimates of single-cell features such as average and total intensity. Numerous shading correction methods have been proposed to remove this effect. In order to compare the performance of different methods, many quantitative performance measures have been developed. However, there is little discussion about which performance measure should be generally applied for evaluation on real data, where the ground truth is absent. In this paper, the state-of-the-art shading correction methods and performance evaluation methods are reviewed. We implement 10 popular shading correction methods on two artificial datasets and four real ones. In order to make an objective comparison between those methods, we employ a number of quantitative performance measures. Extensive validation demonstrates that the coefficient of joint variation (CJV) is the most applicable measure in time-lapse fluorescence images. Based on this measure, we have proposed a novel shading correction method that performs better compared to well-established methods for a range of real data tested. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Near real time, accurate, and sensitive microbiological safety monitoring using an all-fibre spectroscopic fluorescence system

NASA Astrophysics Data System (ADS)

Vanholsbeeck, F.; Swift, S.; Cheng, M.; Bogomolny, E.

2013-11-01

Enumeration of microorganisms is an essential microbiological task for many industrial sectors and research fields. Various tests for detection and counting of microorganisms are used today. However most of the current methods to enumerate bacteria require either long incubation time for limited accuracy, or use complicated protocols along with bulky equipment. We have developed an accurate, all-fibre spectroscopic system to measure fluorescence signal in-situ. In this paper, we examine the potential of this setup for near real time bacteria enumeration in aquatic environment. The concept is based on a well-known phenomenon that the fluorescence quantum yields of some nucleic acid stains significantly increase upon binding with nucleic acids of microorganisms. In addition we have used GFP labeled organisms. The fluorescence signal increase can be correlated to the amount of nucleic acid present in the sample. In addition we have used GFP labeled organisms. Our results show that we are able to detect a wide range of bacteria concentrations without dilution or filtration (1-108 CFU/ml) using different optical probes we designed. This high sensitivity is due to efficient light delivery with an appropriate collection volume and in situ fluorescence detection as well as the use of a sensitive CCD spectrometer. By monitoring the laser power, we can account for laser fluctuations while measuring the fluorescence signal which improves as well the system accuracy. A synchronized laser shutter allows us to achieve a high SNR with minimal integration time, thereby reducing the photobleaching effect. In summary, we conclude that our optical setup may offer a robust method for near real time bacterial detection in aquatic environment.

The detection of T-Nos, a genetic element present in GMOs, by cross-priming isothermal amplification with real-time fluorescence.

PubMed

Zhang, Fang; Wang, Liu; Fan, Kai; Wu, Jian; Ying, Yibin

2014-05-01

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06 × 10(3) copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

Real-time endoscopic guidance using near-infrared fluorescent light for thoracic surgery

NASA Astrophysics Data System (ADS)

Venugopal, Vivek; Stockdale, Alan; Neacsu, Florin; Kettenring, Frank; Frangioni, John V.; Gangadharan, Sidharta P.; Gioux, Sylvain

2013-03-01

Lung cancer is the leading cause of cancer death in the United States, accounting for 28% of all cancer deaths. Standard of care for potentially curable lung cancer involves preoperative radiographic or invasive staging, followed by surgical resection. With recent adjuvant chemotherapy and radiation studies showing a survival advantage in nodepositive patients, it is crucial to accurately stage these patients surgically in order to identify those who may benefit. However, lymphadenectomy in lung cancer is currently performed without guidance, mainly due to the lack of tools permitting real-time, intraoperative identification of lymph nodes. In this study we report the design and validation of a novel, clinically compatible near-infrared (NIR) fluorescence thoracoscope for real-time intraoperative guidance during lymphadenectomy. A novel, NIR-compatible, clinical rigid endoscope has been designed and fabricated, and coupled to a custom source and a dual channel camera to provide simultaneous color and NIR fluorescence information to the surgeon. The device has been successfully used in conjunction with a safe, FDA-approved fluorescent tracer to detect and resect mediastinal lymph nodes during thoracic surgery on Yorkshire pigs. Taken together, this study lays the foundation for the clinical translation of endoscopic NIR fluorescence intraoperative guidance and has the potential to profoundly impact the management of lung cancer patients.

Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification.

PubMed

Guo, Xu-Guang; Zhou, Yong-Zhuo; Li, Qin; Wang, Wei; Wen, Jin-Zhou; Zheng, Lei; Wang, Qian

2018-04-18

To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real-time fluorescence reverse transcription loop-mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with Bst DNA polymerase following real-time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real-time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real-time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

PubMed

Hasan, Md Mehedi; Alam, Mohammad Wajih; Wahid, Khan A; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size.

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability

PubMed Central

Hasan, Md. Mehedi; Wahid, Khan A.; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size. PMID:27977709

Real-Time Tracking the Synthesis and Degradation of Albumin in Complex Biological Systems with a near-Infrared Fluorescent Probe.

PubMed

Jin, Qiang; Feng, Lei; Zhang, Shui-Jun; Wang, Dan-Dan; Wang, Fang-Jun; Zhang, Yi; Cui, Jing-Nan; Guo, Wen-Zhi; Ge, Guang-Bo; Yang, Ling

2017-09-19

In this study, a novel fluorescent detection system for biological sensing of human albumin (HA) was developed on the basis of the pseudoesterase activity and substrate preference of HA. The designed near-infrared (NIR) fluorescent probe (DDAP) could be effectively hydrolyzed by HA, accompanied by significant changes in both color and fluorescence spectrum. The sensing mechanism was fully investigated by fluorescence spectroscopy, NMR, and mass spectra. DDAP exhibited excellent selectivity and sensitivity toward HA over a variety of human plasma proteins, hydrolases, and abundant biomolecules found in human body. The probe has been successfully applied to measure native HA in diluted plasma samples and the secreted HA in the hepatocyte culture supernatant. DDAP has also been used for fluorescence imaging of HA reabsorption in living renal cells, and the results show that the probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. Furthermore, DDAP has been successfully used for real-time tracking the uptaking and degradation of albumin in ex vivo mouse kidney models for the first time. All these results clearly demonstrated that DDAP-based assay held great promise for real-time sensing and tracking HA in complex biological systems, which would be very useful for basic researches and clinical diagnosis of HA-associated diseases.

BODIPY-Based Two-Photon Fluorescent Probe for Real-Time Monitoring of Lysosomal Viscosity with Fluorescence Lifetime Imaging Microscopy.

PubMed

Li, Ling-Ling; Li, Kun; Li, Meng-Yang; Shi, Lei; Liu, Yan-Hong; Zhang, Hong; Pan, Sheng-Lin; Wang, Nan; Zhou, Qian; Yu, Xiao-Qi

2018-05-01

The viscosity of lysosome is reported to be a key indicator of lysosomal functionality. However, the existing mechanical methods of viscosity measurement can hardly be applied at the cellular or subcellular level. Herein, a BODIPY-based two-photon fluorescent probe was presented for monitoring lysosomal viscosity with high spatial and temporal resolution. By installing two morpholine moieties to the fluorophore as target and rotational groups, the TICT effect between the two morpholine rings and the main fluorophore scaffold endowed the probe with excellent viscosity sensitivity. Moreover, Lyso-B succeeded in showing the impact of dexamethasone on lysosomal viscosity in real time.

Fluorescence probes for real-time remote cyanobacteria monitoring: A review of challenges and opportunities.

PubMed

Bertone, Edoardo; Burford, Michele A; Hamilton, David P

2018-05-10

In recent years, there has been a widespread deployment of submersible fluorescence sensors by water utilities. They are used to measure diagnostic pigments and estimate algae and cyanobacteria abundance in near real-time. Despite being useful and promising tools, operators and decision-makers often rely on the data provided by these probes without a full understanding of their limitations. As a result, this may lead to wrong and misleading estimations which, in turn, means that researchers and technicians distrust these sensors. In this review paper, we list and discuss the main limitations of such probes, as well as identifying the effect of environmental factors on pigment production, and in turn, the conversion to cyanobacteria abundance estimation. We argue that a comprehensive calibration approach to obtain reliable readings goes well beyond manufacturers' recommendations, and should involve several context-specific experiments. We also believe that if such a comprehensive set of experiments is conducted, the data collected from fluorescence sensors could be used in artificial intelligence modelling approaches to reliably predict, in near real-time, the presence and abundance of different cyanobacteria species. This would have significant benefits for both drinking and recreational water management, given that cyanobacterial toxicity, and taste and odour compounds production, are species-dependent. Copyright © 2018 Elsevier Ltd. All rights reserved.

Assessment of Spectroscopic, Real-time Ion Thruster Grid Erosion-rate Measurements

NASA Technical Reports Server (NTRS)

Domonkos, Matthew T.; Stevens, Richard E.

2000-01-01

The success of the ion thruster on the Deep Space One mission has opened the gate to the use of primary ion propulsion. Many of the projected planetary missions require throughput and specific impulse beyond those qualified to date. Spectroscopic, real-time ion thruster grid erosion-rate measurements are currently in development at the NASA Glenn Research Center. A preliminary investigation of the emission spectra from an NSTAR derivative thruster with titanium grid was conducted. Some titanium lines were observed in the discharge chamber; however, the signals were too weak to estimate the erosion of the screen grid. Nevertheless, this technique appears to be the only non-intrusive real-time means to evaluate screen grid erosion, and improvement of the collection optics is proposed. Direct examination of the erosion species using laser-induced fluorescence (LIF) was determined to be the best method for a real-time accelerator grid erosion diagnostic. An approach for a quantitative LIF diagnostic was presented.

Real-time Fluorescence Polarization Microscopy of the Moving Boundary in Cross-Gradient SDS-PAGE

NASA Astrophysics Data System (ADS)

Hwang, Jeeseong; Giulian, Gary

2003-03-01

Real-time Fluorescence Polarization Microscopy of the Moving Boundary in Cross-Gradient SDS-PAGE Jeeseong Hwang, Jeffrey R. Krogmeier, Angela M. Bardo, Scott N. Goldie, Lori S. Goldner; Optical Technology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899 Gary G. Giulian, Carl R. Merril; National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a popular method to separate proteins by their apparent molecular weight. However, it is a limited technique due, in part, to its low spatial resolution. In order to improve the resolution and to enhance the detection sensitivity of proteins separated by SDS-PAGE we are studying the detergent properties at the moving boundary of precast Tris-Tricine-Acetate cross-gradient gels using fluorescent cationic and pH indicating dyes. We have developed real-time full-field fluorescence polarization microscopy to monitor the dynamic fluorescence anisotropy from the cationic tetramethylindocarbocyanine dyes localized in the "extended stack", a concentrated detergent zone. We will present quantitative results of the fluorescence anisotropy. Our system is capable of analyzing local structures of the detergent molecules in the moving boundary of SDS-PAGE and the microenvironment(s) near the boundary. We will discuss the significance of these results and their potential role in enhanced protein separation.

Aerosol-fluorescence spectrum analyzer: real-time measurement of emission spectra of airborne biological particles

NASA Astrophysics Data System (ADS)

Hill, Steven C.; Pinnick, Ronald G.; Nachman, Paul; Chen, Gang; Chang, Richard K.; Mayo, Michael W.; Fernandez, Gilbert L.

1995-10-01

We have assembled an aerosol-fluorescence spectrum analyzer (AFS), which can measure the fluorescence spectra and elastic scattering of airborne particles as they flow through a laser beam. The aerosols traverse a scattering cell where they are illuminated with intense (50 kW/cm 2) light inside the cavity of an argon-ion laser operating at 488 nm. This AFS can obtain fluorescence spectra of individual dye-doped polystyrene microspheres as small as 0.5 mu m in diameter. The spectra obtained from microspheres doped with pink and green-yellow dyes are clearly different. We have also detected the fluorescence spectra of airborne particles (although not single particles) made from various

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method

PubMed Central

Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae

2016-01-01

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection. PMID:28018724

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids.

PubMed

Hill, David R; Huang, Sha; Tsai, Yu-Hwai; Spence, Jason R; Young, Vincent B

2017-12-18

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

Real-time monitoring of river water quality using in-line continuous acquisition of fluorescence excitation and emission matrices

NASA Astrophysics Data System (ADS)

Carstea, E.; Baker, A.; Johnson, R.; Reynolds, D. M.

2009-12-01

In-line fluorescence EEM monitoring has been performed over an eleven-day period for Bournbrook River, Birmingham, UK. River water was diverted to a portable laboratory via a continuous flow pump and filter system. Fluorescence excitation-emission matrices data was recorded every 3 minutes using a flow cell (1cm pathlength) coupled to a fiber optic probe. This real-time fluorescence EEM data (Excitation, 225-400 nm at 5 nm steps, emission, 280-500 nm at 2 nm steps) was collected 'in-line'and directly compared with the spectrophotometric properties and physical and chemical parameters of river water samples collected off-line at known time intervals. Over the monitoring period, minor pollution pulses from cross connections were detected and identified hourly along with a random diesel pollution event. This work addresses the practicalities of measuring and detecting fluorescence EEM in the field and discusses the potential of this technological approach for further understanding important hydrological and biogeochemical processes. Problems associated with fouling and system failure are also reported. Example of the data generated from the continuous fluorescence EEM monitoring.

Integrated micro-optofluidic platform for real-time detection of airborne microorganisms

NASA Astrophysics Data System (ADS)

Choi, Jeongan; Kang, Miran; Jung, Jae Hee

2015-11-01

We demonstrate an integrated micro-optofluidic platform for real-time, continuous detection and quantification of airborne microorganisms. Measurements of the fluorescence and light scattering from single particles in a microfluidic channel are used to determine the total particle number concentration and the microorganism number concentration in real-time. The system performance is examined by evaluating standard particle measurements with various sample flow rates and the ratios of fluorescent to non-fluorescent particles. To apply this method to real-time detection of airborne microorganisms, airborne Escherichia coli, Bacillus subtilis, and Staphylococcus epidermidis cells were introduced into the micro-optofluidic platform via bioaerosol generation, and a liquid-type particle collection setup was used. We demonstrate successful discrimination of SYTO82-dyed fluorescent bacterial cells from other residue particles in a continuous and real-time manner. In comparison with traditional microscopy cell counting and colony culture methods, this micro-optofluidic platform is not only more accurate in terms of the detection efficiency for airborne microorganisms but it also provides additional information on the total particle number concentration.

Integrated micro-optofluidic platform for real-time detection of airborne microorganisms

PubMed Central

Choi, Jeongan; Kang, Miran; Jung, Jae Hee

2015-01-01

We demonstrate an integrated micro-optofluidic platform for real-time, continuous detection and quantification of airborne microorganisms. Measurements of the fluorescence and light scattering from single particles in a microfluidic channel are used to determine the total particle number concentration and the microorganism number concentration in real-time. The system performance is examined by evaluating standard particle measurements with various sample flow rates and the ratios of fluorescent to non-fluorescent particles. To apply this method to real-time detection of airborne microorganisms, airborne Escherichia coli, Bacillus subtilis, and Staphylococcus epidermidis cells were introduced into the micro-optofluidic platform via bioaerosol generation, and a liquid-type particle collection setup was used. We demonstrate successful discrimination of SYTO82-dyed fluorescent bacterial cells from other residue particles in a continuous and real-time manner. In comparison with traditional microscopy cell counting and colony culture methods, this micro-optofluidic platform is not only more accurate in terms of the detection efficiency for airborne microorganisms but it also provides additional information on the total particle number concentration. PMID:26522006

A Case Study of Upper-Room UVGI in Densely-Occupied Elementary Classrooms by Real-Time Fluorescent Bioaerosol Measurements.

PubMed

Su, Chunxiao; Lau, Josephine; Yu, Fang

2017-01-08

Recently, the requirement to continuously collect bioaerosol samples using shorter response times has called for the use of real-time detection. The decreased cost of this technology makes it available for a wider application than military use, and makes it accessible to pharmaceutical and academic research. In this case study, real-time bioaerosol monitors (RBMs) were applied in elementary school classrooms-a densely occupied environment-along with upper-room ultraviolet germicidal irradiation (UVGI) devices. The classrooms were separated into a UVGI group and a non-UVGI control group. Fluorescent bioaerosol counts (FBCs) were monitored on 20 visiting days over a four-month period. The classroom with upper-room UVGI showed significantly lower concentrations of fine size (<3 μm) and total FBCs than the control classroom during 13 of the 20 visiting days. The results of the study indicate that the upper-room UVGI could be effective in reducing FBCs in the school environment, and RBMs may be applicable in reflecting the transient conditions of the classrooms due to the dynamic activity levels of the students and teachers.

Real-time fluorescence target/background (T/B) ratio calculation in multimodal endoscopy for detecting GI tract cancer

NASA Astrophysics Data System (ADS)

Jiang, Yang; Gong, Yuanzheng; Wang, Thomas D.; Seibel, Eric J.

2017-02-01

Multimodal endoscopy, with fluorescence-labeled probes binding to overexpressed molecular targets, is a promising technology to visualize early-stage cancer. T/B ratio is the quantitative analysis used to correlate fluorescence regions to cancer. Currently, T/B ratio calculation is post-processing and does not provide real-time feedback to the endoscopist. To achieve real-time computer assisted diagnosis (CAD), we establish image processing protocols for calculating T/B ratio and locating high-risk fluorescence regions for guiding biopsy and therapy in Barrett's esophagus (BE) patients. Methods: Chan-Vese algorithm, an active contour model, is used to segment high-risk regions in fluorescence videos. A semi-implicit gradient descent method was applied to minimize the energy function of this algorithm and evolve the segmentation. The surrounding background was then identified using morphology operation. The average T/B ratio was computed and regions of interest were highlighted based on user-selected thresholding. Evaluation was conducted on 50 fluorescence videos acquired from clinical video recordings using a custom multimodal endoscope. Results: With a processing speed of 2 fps on a laptop computer, we obtained accurate segmentation of high-risk regions examined by experts. For each case, the clinical user could optimize target boundary by changing the penalty on area inside the contour. Conclusion: Automatic and real-time procedure of calculating T/B ratio and identifying high-risk regions of early esophageal cancer was developed. Future work will increase processing speed to <5 fps, refine the clinical interface, and apply to additional GI cancers and fluorescence peptides.

Real-time Visualization and Quantification of Retrograde Cardioplegia Delivery using Near Infrared Fluorescent Imaging

PubMed Central

Rangaraj, Aravind T.; Ghanta, Ravi K.; Umakanthan, Ramanan; Soltesz, Edward G.; Laurence, Rita G.; Fox, John; Cohn, Lawrence H.; Bolman, R. M.; Frangioni, John V.; Chen, Frederick Y.

2009-01-01

Background and Aim of the Study Homogeneous delivery of cardioplegia is essential for myocardial protection during cardiac surgery. Presently, there exist no established methods to quantitatively assess cardioplegia distribution intraoperatively and determine when retrograde cardioplegia is required. In this study, we evaluate the feasibility of near infrared (NIR) imaging for real-time visualization of cardioplegia distribution in a porcine model. Methods A portable, intraoperative, real-time NIR imaging system was utilized. NIR fluorescent cardioplegia solution was developed by incorporating indocyanine green (ICG) into crystalloid cardioplegia solution. Real-time NIR imaging was performed while the fluorescent cardioplegia solution was infused via the retrograde route in 5 ex-vivo normal porcine hearts and in 5 ex-vivo porcine hearts status post left anterior descending (LAD) coronary artery ligation. Horizontal cross-sections of the hearts were obtained at proximal, middle, and distal LAD levels. Videodensitometry was performed to quantify distribution of fluorophore content. Results The progressive distribution of cardioplegia was clearly visualized with NIR imaging. Complete visualization of retrograde distribution occurred within 4 minutes of infusion. Videodensitometry revealed that retrograde cardioplegia primarily distributed to the left ventricle and anterior septum. In hearts with LAD ligation, antegrade cardioplegia did not distribute to the anterior left ventricle. This deficiency was compensated for with retrograde cardioplegia supplementation. Conclusions Incorporation of ICG into cardioplegia allows real-time visualization of cardioplegia delivery via NIR imaging. This technology may prove useful in guiding intraoperative decisions pertaining to when retrograde cardioplegia is mandated. PMID:19016995

Real-time visualization and quantification of retrograde cardioplegia delivery using near infrared fluorescent imaging.

PubMed

Rangaraj, Aravind T; Ghanta, Ravi K; Umakanthan, Ramanan; Soltesz, Edward G; Laurence, Rita G; Fox, John; Cohn, Lawrence H; Bolman, R M; Frangioni, John V; Chen, Frederick Y

2008-01-01

Homogeneous delivery of cardioplegia is essential for myocardial protection during cardiac surgery. Presently, there exist no established methods to quantitatively assess cardioplegia distribution intraoperatively and determine when retrograde cardioplegia is required. In this study, we evaluate the feasibility of near infrared (NIR) imaging for real-time visualization of cardioplegia distribution in a porcine model. A portable, intraoperative, real-time NIR imaging system was utilized. NIR fluorescent cardioplegia solution was developed by incorporating indocyanine green (ICG) into crystalloid cardioplegia solution. Real-time NIR imaging was performed while the fluorescent cardioplegia solution was infused via the retrograde route in five ex vivo normal porcine hearts and in five ex vivo porcine hearts status post left anterior descending (LAD) coronary artery ligation. Horizontal cross-sections of the hearts were obtained at proximal, middle, and distal LAD levels. Videodensitometry was performed to quantify distribution of fluorophore content. The progressive distribution of cardioplegia was clearly visualized with NIR imaging. Complete visualization of retrograde distribution occurred within 4 minutes of infusion. Videodensitometry revealed retrograde cardioplegia, primarily distributed to the left ventricle (LV) and anterior septum. In hearts with LAD ligation, antegrade cardioplegia did not distribute to the anterior LV. This deficiency was compensated for with retrograde cardioplegia supplementation. Incorporation of ICG into cardioplegia allows real-time visualization of cardioplegia delivery via NIR imaging. This technology may prove useful in guiding intraoperative decisions pertaining to when retrograde cardioplegia is mandated.

Note: Design of a full photon-timing recorder down to 1-ns resolution for fluorescence fluctuation measurements

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

Nishimura, Goro, E-mail: gnishi@imd.es.hokudai.ac.jp

2015-10-15

A photon timing recorder was realized in a field programmable gate array to capture all timing data of photons on multiple channels with down to a 1-ns resolution and to transfer all data to a host computer in real-time through universal serial bus with more than 10 M events/s transfer rate. The main concept is that photon time series can be regarded as a serial communication data stream. This recorder was successfully applied for simultaneous measurements of fluorescence fluctuation and lifetime of near-infrared dyes in solution. This design is not only limited to the fluorescence fluctuation measurement but also applicablemore » to any kind of photon counting experiments in a nanosecond time range because of the simple and easily modifiable design.« less

Real time detection of ESKAPE pathogens by a nitroreductase-triggered fluorescence turn-on probe.

PubMed

Xu, Shengnan; Wang, Qinghua; Zhang, Qingyang; Zhang, Leilei; Zuo, Limin; Jiang, Jian-Dong; Hu, Hai-Yu

2017-10-18

The identification of bacterial pathogens is the critical first step in conquering infection diseases. A novel turn-on fluorescent probe for the selective sensing of nitroreductase (NTR) activity and its initial applications in rapid, real-time detection and identification of ESKAPE pathogens have been reported.

Real time monitoring of urban surface water quality using a submersible, tryptophan-like fluorescence sensor

NASA Astrophysics Data System (ADS)

Khamis, Kieran; Bradley, Chris; Hannah, David; Stevens, Rob

2014-05-01

Due to the recent development of field-deployable optical sensor technology, continuous quantification and characterization of surface water dissolved organic matter (DOM) is possible now. Tryptophan-like (T1) fluorescence has the potential to be a particularly useful indicator of human influence on water quality as T1 peaks are associated with the input of labial organic carbon (e.g. sewage or farm waste) and its microbial breakdown. Hence, real-time recording of T1 fluorescence could be particular useful for monitoring waste water infrastructure, treatment efficiency and the identification of contamination events at higher temporal resolution than available hitherto. However, an understanding of sensor measurement repeatability/transferability and interaction with environmental parameters (e.g. turbidity) is required. Here, to address this practical knowledge gap, we present results from a rigorous test of a commercially available submersible tryptophan fluorometer (λex 285, λem 350). Sensor performance was first examined in the laboratory by incrementally increasing turbidity under controlled conditions. Further to this the sensor was integrated into a multi-parameter sonde and field tests were undertaken involving: (i) a spatial sampling campaign across a range of surface water sites in the West Midlands, UK; and (ii) collection of high resolution (sub-hourly) samples from an urban stream (Bournbrook, Birmingham, U.K). To determine the ability of the sensor to capture spatiotemporal dynamics of urban waters DOM was characterized for each site or discrete time step using Excitation Emission Matrix spectroscopy and PARAFAC. In both field and laboratory settings fluorescence intensity was attenuated at high turbidity due to suspended particles increasing absorption and light scattering. For the spatial survey, instrument readings were compared to those obtained by a laboratory grade fluorometer (Varian Cary Eclipse) and a strong, linear relationship was apparent

Real-time color measurement using active illuminant

NASA Astrophysics Data System (ADS)

Tominaga, Shoji; Horiuchi, Takahiko; Yoshimura, Akihiko

2010-01-01

This paper proposes a method for real-time color measurement using active illuminant. A synchronous measurement system is constructed by combining a high-speed active spectral light source and a high-speed monochrome camera. The light source is a programmable spectral source which is capable of emitting arbitrary spectrum in high speed. This system is the essential advantage of capturing spectral images without using filters in high frame rates. The new method of real-time colorimetry is different from the traditional method based on the colorimeter or the spectrometers. We project the color-matching functions onto an object surface as spectral illuminants. Then we can obtain the CIE-XYZ tristimulus values directly from the camera outputs at every point on the surface. We describe the principle of our colorimetric technique based on projection of the color-matching functions and the procedure for realizing a real-time measurement system of a moving object. In an experiment, we examine the performance of real-time color measurement for a static object and a moving object.

Compact wearable dual-mode imaging system for real-time fluorescence image-guided surgery.

PubMed

Zhu, Nan; Huang, Chih-Yu; Mondal, Suman; Gao, Shengkui; Huang, Chongyuan; Gruev, Viktor; Achilefu, Samuel; Liang, Rongguang

2015-09-01

A wearable all-plastic imaging system for real-time fluorescence image-guided surgery is presented. The compact size of the system is especially suitable for applications in the operating room. The system consists of a dual-mode imaging system, see-through goggle, autofocusing, and auto-contrast tuning modules. The paper will discuss the system design and demonstrate the system performance.

Compact wearable dual-mode imaging system for real-time fluorescence image-guided surgery

PubMed Central

Zhu, Nan; Huang, Chih-Yu; Mondal, Suman; Gao, Shengkui; Huang, Chongyuan; Gruev, Viktor; Achilefu, Samuel; Liang, Rongguang

2015-01-01

Abstract. A wearable all-plastic imaging system for real-time fluorescence image-guided surgery is presented. The compact size of the system is especially suitable for applications in the operating room. The system consists of a dual-mode imaging system, see-through goggle, autofocusing, and auto-contrast tuning modules. The paper will discuss the system design and demonstrate the system performance. PMID:26358823

Real-time monitoring of endogenous cysteine levels in living cells using a CD-based ratiometric fluorescent nanoprobe.

PubMed

Wang, Hong; Zhang, Peisheng; Tian, Yong; Zhang, Yuan; Yang, Heping; Chen, Shu; Zeng, Rongjin; Long, Yunfei; Chen, Jian

2018-04-30

A simple and readily available fluorescent probe is needed for the real-time monitoring of endogenous cysteine (Cys) levels in living cells, as such a probe could be used to study the role of Cys in related diseases. Herein, we report the first fluorescent probe based on carbon dots (CDs-FITA) for the selective and ratiometric imaging of endogenous Cys in live cells. In this ratiometric fluorescent probe, a fluorescein derivative (FITA) that recognizes Cys is covalently linked to the surfaces of carbon dots (CDs); employing CDs greatly improves the water solubility of the probe. Acrylate on FITA is selectively cleaved by Cys in aqueous solution under mild conditions, leading to a dramatic increase in the fluorescence from fluorescein. The probe therefore allows the highly selective ratiometric fluorescent detection of Cys even in the presence of various interferents. The as-prepared CDs-FITA showed excellent performance when applied to detect Cys in blood serum. In addition, due to its negligible cytotoxicity, the CDs-FITA can also be utilized for the real-time monitoring of endogenous cysteine (Cys) levels in living cells. Graphical abstract Illustration of the CD-based probe for Cys imaging in living cells.

Rapid detection of Streptococcus pneumoniae by real-time fluorescence loop-mediated isothermal amplification

PubMed Central

Guo, Xu-Guang; Zhou, Shan

2014-01-01

Background and aim of study A significant human pathogenic bacterium, Streptococcus pneumoniae was recognized as a major cause of pneumonia, and is the subject of many humoral immunity studies. Diagnosis is generally made based on clinical suspicion along with a positive culture from a sample from virtually any place in the body. But the testing time is too long. This study is to establish a rapid diagnostic method to identification of Streptococcus pneumoniae. Methods Our laboratory has recently developed a new platform called real-amp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of Streptococcus pneumonia. Two pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the Streptococcus pneumoniae. The amplification was carried out at 63 degree Celsius using SYBR Green for 60 minutes with the tube scanner set to collect fluorescence signals. Clinical samples of Streptococcus pneumoniae and other bacteria were used to determine the sensitivity and specificity of the primers by comparing with traditional culture method. Results The new set of primers consistently detected in laboratory-maintained isolates of Streptococcus pneumoniae from our hospital. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting Streptococcus pneumoniae. Conclusions This study demonstrates that the Streptococcus pneumoniae LAMP primers developed here have the ability to accurately detect Streptococcus pneumoniae infections by real-time fluorescence LAMP. PMID:25276360

Real Time Measures of Effectiveness

DOT National Transportation Integrated Search

2003-06-01

This report describes research that is focused on identifying and determining methods for automatically computing measures of effectiveness (MOEs) when supplied with real time information. The MOEs, along with detection devices such as cameras, roadw...

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.

A portable time-domain LED fluorimeter for nanosecond fluorescence lifetime measurements

NASA Astrophysics Data System (ADS)

Wang, Hongtao; Qi, Ying; Mountziaris, T. J.; Salthouse, Christopher D.

2014-05-01

Fluorescence lifetime measurements are becoming increasingly important in chemical and biological research. Time-domain lifetime measurements offer fluorescence multiplexing and improved handling of interferers compared with the frequency-domain technique. In this paper, an all solid-state, filterless, and highly portable light-emitting-diode based time-domain fluorimeter (LED TDF) is reported for the measurement of nanosecond fluorescence lifetimes. LED based excitation provides more wavelengths options compared to laser diode based excitation, but the excitation is less effective due to the uncollimated beam, less optical power, and longer latency in state transition. Pulse triggering and pre-bias techniques were implemented in our LED TDF to improve the peak optical power to over 100 mW. The proposed pulsing circuit achieved an excitation light fall time of less than 2 ns. Electrical resetting technique realized a time-gated photo-detector to remove the interference of the excitation light with fluorescence. These techniques allow the LED fluorimeter to accurately measure the fluorescence lifetime of fluorescein down to concentration of 0.5 μM. In addition, all filters required in traditional instruments are eliminated for the non-attenuated excitation/emission light power. These achievements make the reported device attractive to biochemical laboratories seeking for highly portable lifetime detection devices for developing sensors based on fluorescence lifetime changes. The device was initially validated by measuring the lifetimes of three commercial fluorophores and comparing them with reported lifetime data. It was subsequently used to characterize a ZnSe quantum dot based DNA sensor.

A portable time-domain LED fluorimeter for nanosecond fluorescence lifetime measurements

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

Wang, Hongtao; Salthouse, Christopher D., E-mail: salthouse@ecs.umass.edu; Center for Personalized Health Monitoring, University of Massachusetts, Amherst, Massachusetts 01003

2014-05-15

Fluorescence lifetime measurements are becoming increasingly important in chemical and biological research. Time-domain lifetime measurements offer fluorescence multiplexing and improved handling of interferers compared with the frequency-domain technique. In this paper, an all solid-state, filterless, and highly portable light-emitting-diode based time-domain fluorimeter (LED TDF) is reported for the measurement of nanosecond fluorescence lifetimes. LED based excitation provides more wavelengths options compared to laser diode based excitation, but the excitation is less effective due to the uncollimated beam, less optical power, and longer latency in state transition. Pulse triggering and pre-bias techniques were implemented in our LED TDF to improve themore » peak optical power to over 100 mW. The proposed pulsing circuit achieved an excitation light fall time of less than 2 ns. Electrical resetting technique realized a time-gated photo-detector to remove the interference of the excitation light with fluorescence. These techniques allow the LED fluorimeter to accurately measure the fluorescence lifetime of fluorescein down to concentration of 0.5 μM. In addition, all filters required in traditional instruments are eliminated for the non-attenuated excitation/emission light power. These achievements make the reported device attractive to biochemical laboratories seeking for highly portable lifetime detection devices for developing sensors based on fluorescence lifetime changes. The device was initially validated by measuring the lifetimes of three commercial fluorophores and comparing them with reported lifetime data. It was subsequently used to characterize a ZnSe quantum dot based DNA sensor.« less

Velocity measurements by laser resonance fluorescence. [single atom diffusional motion

NASA Technical Reports Server (NTRS)

She, C. Y.; Fairbank, W. M., Jr.

1980-01-01

The photonburst correlation method was used to detect single atoms in a buffer gas. Real time flow velocity measurements with laser induced resonance fluorescence from single or multiple atoms was demonstrated and this method was investigated as a tool for wind tunnel flow measurement. Investigations show that single atoms and their real time diffusional motion on a buffer gas can be measured by resonance fluorescence. By averaging over many atoms, flow velocities up to 88 m/s were measured in a time of 0.5 sec. It is expected that higher flow speeds can be measured and that the measurement time can be reduced by a factor of 10 or more by careful experimental design. The method is clearly not ready for incorporation in high speed wind tunnels because it is not yet known whether the stray light level will be higher or lower, and it is not known what detection efficiency can be obtained in a wind tunnel situation.

Fluorescence particle detector for real-time quantification of viable organisms in air

NASA Astrophysics Data System (ADS)

Luoma, Greg; Cherrier, Pierre P.; Piccioni, Marc; Tanton, Carol; Herz, Steve; DeFreez, Richard K.; Potter, Michael; Girvin, Kenneth L.; Whitney, Ronald

2002-02-01

The ability to detect viable organisms in air in real time is important in a number of applications. Detecting high levels of airborne organisms in hospitals can prevent post-operative infections and the spread of diseases. Monitoring levels of airborne viable organisms in pharmaceutical facilities can ensure safe production of drugs or vaccines. Monitoring airborne bacterial levels in meat processing plants can help to prevent contamination of food products. Monitoring the level of airborne organisms in bio-containment facilities can ensure that proper procedures are being followed. Finally, detecting viable organisms in real time is a key to defending against biological agent attacks. This presentation describes the development and performance of a detector, based on fluorescence particle counting technology, where an ultraviolet laser is used to count particles by light scattering and elicit fluorescence from specific biomolecules found only in living organisms. The resulting detector can specifically detect airborne particles containing living organisms from among the large majority of other particles normally present in air. Efforts to develop the core sensor technology, focusing on integrating an UV laser with a specially designed particle-counting cell will be highlighted. The hardware/software used to capture the information from the sensor, provide an alarm in the presence of an unusual biological aerosol content will also be described. Finally, results from experiments to test the performance of the detector will be presented.

Real-time measurements of jet aircraft engine exhaust.

PubMed

Rogers, Fred; Arnott, Pat; Zielinska, Barbara; Sagebiel, John; Kelly, Kerry E; Wagner, David; Lighty, JoAnn S; Sarofim, Adel F

2005-05-01

Particulate-phase exhaust properties from two different types of ground-based jet aircraft engines--high-thrust and turboshaft--were studied with real-time instruments on a portable pallet and additional time-integrated sampling devices. The real-time instruments successfully characterized rapidly changing particulate mass, light absorption, and polycyclic aromatic hydrocarbon (PAH) content. The integrated measurements included particulate-size distributions, PAH, and carbon concentrations for an entire test run (i.e., "run-integrated" measurements). In all cases, the particle-size distributions showed single modes peaking at 20-40nm diameter. Measurements of exhaust from high-thrust F404 engines showed relatively low-light absorption compared with exhaust from a turboshaft engine. Particulate-phase PAH measurements generally varied in phase with both net particulate mass and with light-absorbing particulate concentrations. Unexplained response behavior sometimes occurred with the real-time PAH analyzer, although on average the real-time and integrated PAH methods agreed within the same order of magnitude found in earlier investigations.

Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer

PubMed Central

Elliott, Amicia D.; Gao, Liang; Ustione, Alessandro; Bedard, Noah; Kester, Robert; Piston, David W.; Tkaczyk, Tomasz S.

2012-01-01

Summary The development of multi-colored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of a snapshot hyperspectral imaging device, the image mapping spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signal capture from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined fluorescent protein (FP)-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in pancreatic β-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope. PMID:22854044

Real-time method and apparatus for measuring the decay-time constant of a fluorescing phosphor

DOEpatents

Britton, Jr., Charles L.; Beshears, David L.; Simpson, Marc L.; Cates, Michael R.; Allison, Steve W.

1999-01-01

A method for determining the decay-time constant of a fluorescing phosphor is provided, together with an apparatus for performing the method. The apparatus includes a photodetector for detecting light emitted by a phosphor irradiated with an excitation pulse and for converting the detected light into an electrical signal. The apparatus further includes a differentiator for differentiating the electrical signal and a zero-crossing discrimination circuit that outputs a pulse signal having a pulse width corresponding to the time period between the start of the excitation pulse and the time when the differentiated electrical signal reaches zero. The width of the output pulse signal is proportional to the decay-time constant of the phosphor.

Real-time fluorescence imaging of the DNA damage repair response during mitosis.

PubMed

Miwa, Shinji; Yano, Shuya; Yamamoto, Mako; Matsumoto, Yasunori; Uehara, Fuminari; Hiroshima, Yukihiko; Toneri, Makoto; Murakami, Takashi; Kimura, Hiroaki; Hayashi, Katsuhiro; Yamamoto, Norio; Efimova, Elena V; Tsuchiya, Hiroyuki; Hoffman, Robert M

2015-04-01

The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe. © 2014 Wiley Periodicals, Inc.

Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model.

PubMed

Jim, Kin Ki; Engelen-Lee, JooYeon; van der Sar, Astrid M; Bitter, Wilbert; Brouwer, Matthijs C; van der Ende, Arie; Veening, Jan-Willem; van de Beek, Diederik; Vandenbroucke-Grauls, Christina M J E

2016-08-19

Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavourable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin-deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked reduction of phagocytes. This new meningitis model permits detailed analysis and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.

Fluorescence acquisition during hybridization phase in quantitative real-time PCR improves specificity and signal-to-noise ratio.

PubMed

Mehndiratta, Mohit; Palanichamy, Jayanth Kumar; Ramalingam, Pradeep; Pal, Arnab; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2008-12-01

Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.

Gold nanoclusters-Cu(2+) ensemble-based fluorescence turn-on and real-time assay for acetylcholinesterase activity and inhibitor screening.

PubMed

Sun, Jian; Yang, Xiurong

2015-12-15

Based on the specific binding of Cu(2+) ions to the 11-mercaptoundecanoic acid (11-MUA)-protected AuNCs with intense orange-red emission, we have proposed and constructed a novel fluorescent nanomaterials-metal ions ensemble at a nonfluorescence off-state. Subsequently, an AuNCs@11-MUA-Cu(2+) ensemble-based fluorescent chemosensor, which is amenable to convenient, sensitive, selective, turn-on and real-time assay of acetylcholinesterase (AChE), could be developed by using acetylthiocholine (ATCh) as the substrate. Herein, the sensing ensemble solution exhibits a marvelous fluorescent enhancement in the presence of AChE and ATCh, where AChE hydrolyzes its active substrate ATCh into thiocholine (TCh), and then TCh captures Cu(2+) from the ensemble, accompanied by the conversion from fluorescence off-state to on-state of the AuNCs. The AChE activity could be detected less than 0.05 mU/mL within a good linear range from 0.05 to 2.5 mU/mL. Our proposed fluorescence assay can be utilized to evaluate the AChE activity quantitatively in real biological sample, and furthermore to screen the inhibitor of AChE. As far as we know, the present study has reported the first analytical proposal for sensing AChE activity in real time by using a fluorescent nanomaterials-Cu(2+) ensemble or focusing on the Cu(2+)-triggered fluorescence quenching/recovery. This strategy paves a new avenue for exploring the biosensing applications of fluorescent AuNCs, and presents the prospect of AuNCs@11-MUA-Cu(2+) ensemble as versatile enzyme activity assay platforms by means of other appropriate substrates/analytes. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of fluorescent tracers for the real-time monitoring of renal function

NASA Astrophysics Data System (ADS)

Poreddy, Amruta R.; Asmelash, Bethel; Debreczeny, Martin P.; Fitch, Richard M.; Freskos, John N.; Galen, Karen P.; Gaston, Kimberly R.; Kostelc, James G.; Kumar, Rana; Marzan, Tim A.; Neumann, William L.; Rajagopalan, Raghavan; Schoenstein, Tasha M.; Shieh, Jeng-Jong; Wilcox, J. Micah; Wojdyla, Jolette K.; Dorshow, Richard B.

2011-03-01

Accurate measurement of glomerular filtration rate (GFR) at the bedside is highly desirable in order to assess renal function in real-time, which is currently an unmet clinical need. In our pursuit to develop exogenous fluorescent tracers as GFR markers, various hydrophilic derivatives of 3,6-diaminopyrazine-2,5-dicarboxylic acid with varying molecular weights and absorption/emission characteristics were synthesized. These include polyhydroxyalkyl based small molecules and poly(ethylene glycol) (PEG) substituted moderate molecular weight compounds, which were further sub-grouped into analogs having blue excitation with green emission, and relatively longer wavelength analogs having green excitation with orange emission. Lead compounds were identified in each of the four classes on the basis of structure- activity relationship studies, which included in vitro plasma protein binding, in vivo urine recovery of administered dose, and in vivo optical monitoring. The in vivo optical monitoring experiments with lead candidates have been correlated with plasma pharmacokinetic (PK) data for measurement of clearance and hence GFR. Renal clearance of these compounds, occurring exclusively via glomerular filtration, was established by probenecid blocking experiments. The renal clearance property of all these advanced candidates was superior to that of the iothalamate, which is currently an accepted standard for the measurement of GFR.

Observation of development of breast cancer cell lines in real time by fluorescence microscopy under simulated microgravity

NASA Astrophysics Data System (ADS)

Lavan, David; Valdivia-Silva, Julio E.; Sanabria, Gabriela; Orihuela, Diego; Suarez, Juan; Quispe, Marco; Chuchon, Mariano; Martin, David; Maroto, Marcos; Egea, Javier

2016-07-01

This project consist in the implementation of a fluorescence microscope for the in real time monitoring of biological labeled samples by several fluorophores in microgravity conditions keeping the temperature, humidity, and (CO)2 controlled by an electronic platform. The system (fluorescence microscope and incubator) is integrated to a microgravity simulator machine which was presented on the "30th Annual American Society for Gravitation and Space Research Meeting" October 2014 in Pasadena, CA, USA. Currently, we have the microgravity machine biologically validated by genetic expression studies in pupal stage of Drosophila melanogaster. The fluorescence microscope has a platform designed to hold a culture flask, and a fluorescence camera (Leica DFC3000 G) connected to an optical system (Fluorescence Light source Leica EL6000, optic fiber, fiber adapter, and fluorescence filter) in order to take images in real time. The mechanical system of the fluorescence microsc ope is designed to allow the displacement of the fluorescence camera through a parallel plane to the culture flask's plane and also the movement of the platform through a perpendicular axis to the culture flask in order to focus the samples to the optical system. The mechanical system is propelled by four DC moto-reductors with encoder (A-max 26 Maxon motor, GP 32S screw and MR encoder) that generate displacements in the order of micrometers. The angular position control of the DC motoreductor's shaft of all the DC moto-reductors is done by PWM signals based on the interpretation of the signals provided by the encoders during the movement. The system is remotely operated by a graphic interface installed on a personal computer or any mobile device (smartphone, laptop or tablet) by using the internet. Acknowledgments: Grant of INNOVATE PERU (Formerly FINCYT)

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

PubMed Central

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

2015-01-01

Abstract. Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures. PMID:26440760

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

PubMed

Watson, Jeffrey R; Gainer, Christian F; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G Michael; Anton, Rein; Romanowski, Marek

2015-10-01

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

NASA Astrophysics Data System (ADS)

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael, Jr.; Anton, Rein; Romanowski, Marek

2015-10-01

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic imaging

NASA Astrophysics Data System (ADS)

Chen, Zhenyue; Deán-Ben, Xosé Luís.; Gottschalk, Sven; Razansky, Daniel

2018-02-01

Fluorescence imaging is widely employed in all fields of cell and molecular biology due to its high sensitivity, high contrast and ease of implementation. However, the low spatial resolution and lack of depth information, especially in strongly-scattering samples, restrict its applicability for deep-tissue imaging applications. On the other hand, optoacoustic imaging is known to deliver a unique set of capabilities such as high spatial and temporal resolution in three dimensions, deep penetration and spectrally-enriched imaging contrast. Since fluorescent substances can generate contrast in both modalities, simultaneous fluorescence and optoacoustic readings can provide new capabilities for functional and molecular imaging of living organisms. Optoacoustic images can further serve as valuable anatomical references based on endogenous hemoglobin contrast. Herein, we propose a hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic tomography, both operating in reflection mode, which synergistically combines the advantages of stand-alone systems. Validation of the spatial resolution and sensitivity of the system were first carried out in tissue mimicking phantoms while in vivo imaging was further demonstrated by tracking perfusion of an optical contrast agent in a mouse brain in the hybrid imaging mode. Experimental results show that the proposed system effectively exploits the contrast mechanisms of both imaging modalities, making it especially useful for accurate monitoring of fluorescence-based signal dynamics in highly scattering samples.

Real-time PCR detection chemistry.

PubMed

Navarro, E; Serrano-Heras, G; Castaño, M J; Solera, J

2015-01-15

Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review. Copyright © 2014 Elsevier B.V. All rights reserved.

Real-time assessment of breast surgical margins with fluorescence-guided microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor V.; Park, Jesung; Maguluri, Gopi N.; Krishnamurthy, Savitri

2017-02-01

A novel multimodal optical imaging approach for real-time assessment of surgical margins on breast cancer lumpectomy specimens is presented. Our approach is to target cancer cells using an optically silent peptide substrate containing two (NIR) fluorochromes, internally quenched, which are cleaved by highly expressed breast cancer enzymes, like urokinase-type plasminogen activator (uPA). Thus this agent becomes highly fluorescent only on the cancer area when the specimen is excited by a NIR laser beam. A fluorescence imager is used to highlight cancer-suspect margins on the surgical specimen, while high-resolution optical coherence tomography (OCT) imaging is used to visualize tissue morphology on the highlighted areas and confirm or rule out cancer presence. This technology will hopefully increase the success rate of cancer surgeries, reduce the risk of cancer recurrence and significantly reduce US healthcare costs.

Real-time method and apparatus for measuring the temperature of a fluorescing phosphor

DOEpatents

Britton, Jr., Charles L.; Beshears, David L.; Simpson, Marc L.; Cates, Michael R.; Allison, Steve W.

1999-01-01

A method for determining the temperature of a fluorescing phosphor is provided, together with an apparatus for performing the method. The apparatus includes a photodetector for detecting light emitted by a phosphor irradiated with an excitation pulse and for converting the detected light into an electrical signal. The apparatus further includes a differentiator for differentiating the electrical signal and a zero-crossing discrimination circuit that outputs a pulse signal having a pulse width corresponding to the time period between the start of the excitation pulse and the time when the differentiated electrical signal reaches zero. The width of the output pulse signal is proportional to the decay-time constant of the phosphor.

Fluorescent probes for real-time measurement of nitric oxide in living cells.

PubMed

Li, Huili; Wan, Ajun

2015-11-07

Nitric oxide (NO) is an important signaling molecule in biology. Both NO excess and insufficiency have been implicated in numerous physiological and pathological conditions. In order to study the diverse biological roles of NO in cells and tissues, many techniques have been developed for assaying NO. Recently, new generations of fluorescent probes have become indispensible tools for the study of NO biology because of their sensitivity, selectivity, spatiotemporal resolution, and experimental feasibility. Rational application of these probes in the study requires the understanding of the molecular mechanism that the probes are involved in. In this review, we will present an arsenal of fluorescent probes used to detect NO in living cells and animal tissues. We will also discuss the molecular mechanisms, actualities and prospects of fluorescent probes in detecting NO in cell biology.

Visual in vivo degradation of injectable hydrogel by real-time and non-invasive tracking using carbon nanodots as fluorescent indicator.

PubMed

Wang, Lei; Li, Baoqiang; Xu, Feng; Li, Ying; Xu, Zheheng; Wei, Daqing; Feng, Yujie; Wang, Yaming; Jia, Dechang; Zhou, Yu

2017-11-01

Visual in vivo degradation of hydrogel by fluorescence-related tracking and monitoring is crucial for quantitatively depicting the degradation profile of hydrogel in a real-time and non-invasive manner. However, the commonly used fluorescent imaging usually encounters limitations, such as intrinsic photobleaching of organic fluorophores and uncertain perturbation of degradation induced by the change in molecular structure of hydrogel. To address these problems, we employed photoluminescent carbon nanodots (CNDs) with low photobleaching, red emission and good biocompatibility as fluorescent indicator for real-time and non-invasive visual in vitro/in vivo degradation of injectable hydrogels that are mixed with CNDs. The in vitro/in vivo toxicity results suggested that CNDs were nontoxic. The embedded CNDs in hydrogels did not diffuse outside in the absence of hydrogel degradation. We had acquired similar degradation kinetics (PBS-Enzyme) between gravimetric and visual determination, and established mathematical equation to quantitatively depict in vitro degradation profile of hydrogels for the predication of in vivo hydrogel degradation. Based on the in vitro data, we developed a visual platform that could quantitatively depict in vivo degradation behavior of new injectable biomaterials by real-time and non-invasive fluorescence tracking. This fluorescence-related visual imaging methodology could be applied to subcutaneous degradation of injectable hydrogel with down to 7 mm depth in small animal trials so far. This fluorescence-related visual imaging methodology holds great potentials for rational design and convenient in vivo screening of biocompatible and biodegradable injectable hydrogels in tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.

Smartphone Cortex Controlled Real-Time Image Processing and Reprocessing for Concentration Independent LED Induced Fluorescence Detection in Capillary Electrophoresis.

PubMed

Szarka, Mate; Guttman, Andras

2017-10-17

We present the application of a smartphone anatomy based technology in the field of liquid phase bioseparations, particularly in capillary electrophoresis. A simple capillary electrophoresis system was built with LED induced fluorescence detection and a credit card sized minicomputer to prove the concept of real time fluorescent imaging (zone adjustable time-lapse fluorescence image processor) and separation controller. The system was evaluated by analyzing under- and overloaded aminopyrenetrisulfonate (APTS)-labeled oligosaccharide samples. The open source software based image processing tool allowed undistorted signal modulation (reprocessing) if the signal was inappropriate for the actual detection system settings (too low or too high). The novel smart detection tool for fluorescently labeled biomolecules greatly expands dynamic range and enables retrospective correction for injections with unsuitable signal levels without the necessity to repeat the analysis.

Intraoperative fluorescence-based enhanced reality laparoscopic real-time imaging to assess bowel perfusion at the anastomotic site in an experimental model.

PubMed

Diana, M; Agnus, V; Halvax, P; Liu, Y-Y; Dallemagne, B; Schlagowski, A-I; Geny, B; Diemunsch, P; Lindner, V; Marescaux, J

2015-01-01

Fluorescence videography is a promising technique for assessing bowel perfusion. Fluorescence-based enhanced reality (FLER) is a novel concept, in which a dynamic perfusion cartogram, generated by computer analysis, is superimposed on to real-time laparoscopic images. The aim of this experimental study was to assess the accuracy of FLER in detecting differences in perfusion in a small bowel resection-anastomosis model. A small bowel ischaemic segment was created laparoscopically in 13 pigs. Animals were allocated to having anastomoses performed at either low perfusion (25 per cent; n = 7) or high perfusion (75 per cent; n = 6), as determined by FLER analysis. Capillary lactate levels were measured in blood samples obtained by serosal puncturing in the ischaemic area, resection lines and vascularized areas. Pathological inflammation scoring of the anastomosis was carried out. Lactate levels in the ischaemic area (mean(s.d.) 5·6(2·8) mmol/l) were higher than those in resection lines at 25 per cent perfusion (3·7(1·7) mmol/l; P = 0·010) and 75 per cent perfusion (2·9(1·3) mmol/l; P < 0·001), and higher than levels in vascular zones (2·5(1·0) mmol/l; P < 0·001). Lactate levels in resection lines with 75 per cent perfusion were lower than those in lines with 25 per cent perfusion (P < 0·001), and similar to those in vascular zones (P = 0·188). Levels at resection lines with 25 per cent perfusion were higher than those in vascular zones (P = 0·001). Mean(s.d.) global inflammation scores were higher in the 25 per cent perfusion group compared with the 75 per cent perfusion group for mucosa/submucosa (2·1(0·4) versus 1·2(0·4); P = 0·003) and serosa (1·8(0·4) versus 0·8(0·8); P = 0·014). A ratio of preanastomotic lactate levels in the ischaemic area relative to the resection lines of 2 or less was predictive of a more severe inflammation score. In an experimental model, FLER appeared accurate in discriminating bowel perfusion levels. Surgical

Tumor detection in mice by measurement of fluorescence decay time matrices

NASA Astrophysics Data System (ADS)

Cubeddu, R.; Pifferi, A.; Taroni, P.; Valentini, G.; Canti, G.

1995-12-01

An intensified CCD video camera has been used to measure the spatial distribution of the fluorescence decay time in tumor-bearing mice sensitized with hematoporphyrin derivative. Mice were injected with five doses of sensitizer, ranging from 0.1 to 10 mg / kg body weight. For any drug dose the decay time of the exogenous fluorescence in the tumor is always significantly longer than in normal tissues. The image created by associating a gray-shade scale to the decay time matrix of each mouse permits a reliable and precise detection of the neoplasia.

Fluorescence Spectra of Individual Flowing Airborne Biological Particles Measured in Real Time

DTIC Science & Technology

2001-02-01

and fungal spores ( Aspergillus versicolor, ATCC 9577). B. subtilis var. niger (lyophilized cells) and E. herbicola were grown by streak- ing onto...Excitation Figure 7 shows fluorescence spectra of B. subtilis var. niger vegetative cells and fungal spores ( Aspergillus versicolor), both 5 µm in diameter...µm-diam clusters of B. subtilis var. niger spores, and B. subtilis var. niger vegetative cells ……………………………………… 10 5. Fluorescence spectra of starved

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria.

PubMed

Lucchi, Naomi W; Demas, Allison; Narayanan, Jothikumar; Sumari, Deborah; Kabanywanyi, Abdunoor; Kachur, S Patrick; Barnwell, John W; Udhayakumar, Venkatachalam

2010-10-29

Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.

Real-time Measurements of Biological Particles at Several Continental Sites using the WIBS-4A

NASA Astrophysics Data System (ADS)

McMeeking, G. R.; Kok, G. L.; Petters, M. D.; Wright, T.; Hader, J.; Mccubbin, I. B.; Hallar, A. G.; Twohy, C. H.; Toohey, D. W.; DeMott, P. J.; McCluskey, C.; Baumgardner, D.

2013-12-01

Biological particles (bacteria, fungi/fungal spores, viruses, algae and fragments of biological material) may play a significant role in modifying cloud properties by acting as ice nuclei and thus have an indirect effect on climate forcing. Little is known, however, regarding the abundance and distribution of biological particles and their importance to cloud microphysics in different environments. On-line, continuous measurement systems offer the potential to measure biological systems at high time resolution and sensitivity, providing greater insight into their distribution in the atmosphere, dispersal mechanisms and potential soures. The WIBS-4A (Wideband Integrated Bioaerosol Sensor) detects fluorescent biological material in real-time associated with individual particles. It measures five properties: a) optical size via light scattering, b) fluorescent emissions in the wavelength range 310-400 following excitation by 280 nm light, c) fluorescent emissions in the wavelength range 420-650 following excitation by 280 nm light, d) fluorescent emissions in the wavelength range 420-650 following excitation by 370 nm light, and e) particle asymmetry factor based on intensities of forward scattered light onto a 4-element detector. Together, these properties aid the classification of sampled particles that contain biofluorophores such as tryptophan or NAD(P)H, which can be found in biological particles. Here we present results from a series of laboratory, ground- and aircraft-based measurements of biological particles using the WIBS-4A. The studies include airborne measurements over the United States, ground-based measurements at a coastal site, an urban site in the southeast US and a high alpine site, and laboratory measurements of a variety of biological and non-biological particles. Our analysis focused on both the characterization of the instrument response as well as an evaluation of its suitability for performing ambient measurements and potential artifacts. We

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Time-resolved UV-excited microarray reader for fluorescence energy transfer (FRET) measurements

NASA Astrophysics Data System (ADS)

Orellana, Adelina; Hokkanen, Ari P.; Pastinen, Tomi; Takkinen, Kristina; Soderlund, Hans

2001-05-01

Analytical systems based on immunochemistry are largely used in medical diagnostics and in biotechnology. There is a significant pressure to develop the present assay formats to become easier to use, faster, and less reagent consuming. Further developments towards high density array--like multianalyte measurement systems would be valuable. To this aim we have studied the applicability of fluorescence resonance energy transfer and time-resolved fluorescence resonance energy transfer in immunoassays on microspots and in microwells. We have used engineered recombinant antibodies detecting the pentameric protein CRP as a model analyte system, and tested different assay formats. We describe also the construction of a time-resolved scanning epifluorometer with which we could measure the FRET interaction between the slow fluorescence decay from europium chelates and its energy transfer to the rapidly decaying fluorophore Cy5.

Real-time monitoring of anticancer drug release with highly fluorescent star-conjugated copolymer as a drug carrier.

PubMed

Qiu, Feng; Wang, Dali; Zhu, Qi; Zhu, Lijuan; Tong, Gangsheng; Lu, Yunfeng; Yan, Deyue; Zhu, Xinyuan

2014-04-14

Chemotherapy is one of the major systemic treatments for cancer, in which the drug release kinetics is a key factor for drug delivery. In the present work, a versatile fluorescence-based real-time monitoring system for intracellular drug release has been developed. First, two kinds of star-conjugated copolymers with different connections (e.g., pH-responsive acylhydrazone and stable ether) between a hyperbranched conjugated polymer (HCP) core and many linear poly(ethylene glycol) (PEG) arms were synthesized. Owing to the amphiphilic three-dimensional architecture, the star-conjugated copolymers could self-assemble into multimicelle aggregates from unimolecular micelles with excellent emission performance in the aqueous medium. When doxorubicin (DOX) as a model drug was encapsulated into copolymer micelles, the emission of star-conjugated copolymer and DOX was quenched. In vitro biological studies revealed that fluorescent intensities of both star-conjugated copolymer and DOX were activated when the drug was released from copolymeric micelles, resulting in the enhanced cellular proliferation inhibition against cancer cells. Importantly, pH-responsive feature of the star-conjugated copolymer with acylhydrazone linkage exhibited accelerated DOX release at a mildly acidic environment, because of the fast breakage of acylhydrazone in endosome or lysosome of tumor cells. Such fluorescent star-conjugated copolymers may open up new perspectives to real-time study of drug release kinetics of polymeric drug delivery systems for cancer therapy.

Real-time hostile attribution measurement and aggression in children.

PubMed

Yaros, Anna; Lochman, John E; Rosenbaum, Jill; Jimenez-Camargo, Luis Alberto

2014-01-01

Hostile attributions are an important predictor of aggression in children, but few studies have measured hostile attributions as they occur in real-time. The current study uses an interactive video racing game to measure hostile attributions while children played against a presumed peer. A sample of 75 children, ages 10-13, used nonverbal and verbal procedures to respond to ambiguous provocation by their opponent. Hostile attributions were significantly positively related to parent-rated reactive aggression, when controlling for proactive aggression. Hostile attributions using a nonverbal response procedure were negatively related to proactive aggression, when controlling for reactive aggression. Results suggest hostile attributions in real-time occur quickly and simultaneously with social interaction, which differs from the deliberative, controlled appraisals measured with vignette-based instruments. The relation between real-time hostile attributions and reactive aggression could be accounted for by the impulsive response style that is characteristic of reactive aggression, whereas children exhibiting proactive aggression may be more deliberate and intentional in their responding, resulting in a negative relation with real-time hostile attributions. These findings can be used both to identify children at risk for aggression and to enhance preventive interventions. © 2014 Wiley Periodicals, Inc.

The Potential Applications of Real-Time Monitoring of Water Quality in a Large Shallow Lake (Lake Taihu, China) Using a Chromophoric Dissolved Organic Matter Fluorescence Sensor

PubMed Central

Niu, Cheng; Zhang, Yunlin; Zhou, Yongqiang; Shi, Kun; Liu, Xiaohan; Qin, Boqiang

2014-01-01

This study presents results from field surveys performed over various seasons in a large, eutrophic, shallow lake (Lake Taihu, China) using an in situ chromophoric dissolved organic matter (CDOM) fluorescence sensor as a surrogate for other water quality parameters. These measurements identified highly significant empirical relationships between CDOM concentration measured using the in situ fluorescence sensor and CDOM absorption, fluorescence, dissolved organic carbon (DOC), chemical oxygen demand (COD) and total phosphorus (TP) concentrations. CDOM concentration expressed in quinine sulfate equivalent units, was highly correlated with the CDOM absorption coefficient (r2 = 0.80, p < 0.001), fluorescence intensities (Ex./Em. 370/460 nm) (r2 = 0.91, p < 0.001), the fluorescence index (r2 = 0.88, p < 0.001) and the humification index (r2 = 0.78, p < 0.001), suggesting that CDOM concentration measured using the in situ fluorescence sensor could act as a substitute for the CDOM absorption coefficient and fluorescence measured in the laboratory. Similarly, CDOM concentration was highly correlated with DOC concentration (r2 = 0.68, p < 0.001), indicating that in situ CDOM fluorescence sensor measurements could be a proxy for DOC concentration. In addition, significant positive correlations were found between laboratory CDOM absorption coefficients and COD (r2 = 0.83, p < 0.001), TP (r2 = 0.82, p < 0.001) concentrations, suggesting a potential further application for the real-time monitoring of water quality using an in situ CDOM fluorescence sensor. PMID:24984060

The potential applications of real-time monitoring of water quality in a large shallow lake (Lake Taihu, China) using a chromophoric dissolved organic matter fluorescence sensor.

PubMed

Niu, Cheng; Zhang, Yunlin; Zhou, Yongqiang; Shi, Kun; Liu, Xiaohan; Qin, Boqiang

2014-06-30

This study presents results from field surveys performed over various seasons in a large, eutrophic, shallow lake (Lake Taihu, China) using an in situ chromophoric dissolved organic matter (CDOM) fluorescence sensor as a surrogate for other water quality parameters. These measurements identified highly significant empirical relationships between CDOM concentration measured using the in situ fluorescence sensor and CDOM absorption, fluorescence, dissolved organic carbon (DOC), chemical oxygen demand (COD) and total phosphorus (TP) concentrations. CDOM concentration expressed in quinine sulfate equivalent units, was highly correlated with the CDOM absorption coefficient (r(2) = 0.80, p < 0.001), fluorescence intensities (Ex./Em. 370/460 nm) (r(2) = 0.91, p < 0.001), the fluorescence index (r(2) = 0.88, p < 0.001) and the humification index (r(2) = 0.78, p < 0.001), suggesting that CDOM concentration measured using the in situ fluorescence sensor could act as a substitute for the CDOM absorption coefficient and fluorescence measured in the laboratory. Similarly, CDOM concentration was highly correlated with DOC concentration (r(2) = 0.68, p < 0.001), indicating that in situ CDOM fluorescence sensor measurements could be a proxy for DOC concentration. In addition, significant positive correlations were found between laboratory CDOM absorption coefficients and COD (r(2) = 0.83, p < 0.001), TP (r(2) = 0.82, p < 0.001) concentrations, suggesting a potential further application for the real-time monitoring of water quality using an in situ CDOM fluorescence sensor.

Quantitative real-time imaging of glutathione

USDA-ARS?s Scientific Manuscript database

Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time ...

Real-Time PCR (qPCR) Primer Design Using Free Online Software

ERIC Educational Resources Information Center

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid*

PubMed Central

Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

2014-01-01

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m−1 s−1) were significantly higher than that measured for H2O2 (1.5 m−1 s−1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 103 m−1 s−1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. PMID:24928516

Fluorescent nanosensors for intracellular measurements: synthesis, characterization, calibration, and measurement

PubMed Central

Desai, Arpan S.; Chauhan, Veeren M.; Johnston, Angus P. R.; Esler, Tim; Aylott, Jonathan W.

2013-01-01

Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore, nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer) and a pH-insensitive reference fluorophore (internal standard) immobilized in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesized using standard laboratory equipment and are detectable by non-invasive widely accessible imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular, special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: (1) synthesis and characterization of polyacrylamide and silica based nanosensors, (2) nanosensor calibration and (3) performing measurements using fluorescence microscopy. PMID:24474936

Real-time PCR (qPCR) primer design using free online software.

PubMed

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

Real-time measurement of UV-inactivated Escherichia coli bacterial particles by electrospray-assisted UVAPS spectrometry.

PubMed

Jung, Jae Hee; Lee, Jung Eun; Bae, Gwi Nam

2011-08-01

The ultraviolet aerodynamic particle sizer (UVAPS) is a novel commercially available aerosol spectrometer for real-time continuous monitoring of viable bioaerosols, based on fluorescence from living microorganisms. In a previous study, we developed an electrospray-assisted UVAPS using biological electrospray techniques, which have the advantage of generating non-agglomerated single particles by the repulsive electrical forces. With this electrospraying of suspensions containing microorganisms, the analytical system can supply more accurate and quantitative information about living microorganisms than with conventional aerosolization. Using electrospray-assisted UVAPS, we investigated the characteristics of bacterial particles with various viabilities in real-time. Escherichia coli was used as the test microorganism, and its initial viability was controlled by the degree of exposure to UV irradiation. In the stable cone-jet domain, the particle size distributions of test bacterial particles remained almost uniform regardless of the degree of UV inactivation. However, the fluorescence spectra of the bacterial particles changed with the degree of UV inactivation. The fluorescence characteristics of UV-inactivated bacterial particles tended to show a similar decline with viability, determined by the sampling and culture method, although the percentage showing fluorescence was higher than that showing viability. Copyright © 2011 Elsevier B.V. All rights reserved.

Real-time autocorrelator for fluorescence correlation spectroscopy based on graphical-processor-unit architecture: method, implementation, and comparative studies

NASA Astrophysics Data System (ADS)

Laracuente, Nicholas; Grossman, Carl

2013-03-01

We developed an algorithm and software to calculate autocorrelation functions from real-time photon-counting data using the fast, parallel capabilities of graphical processor units (GPUs). Recent developments in hardware and software have allowed for general purpose computing with inexpensive GPU hardware. These devices are more suited for emulating hardware autocorrelators than traditional CPU-based software applications by emphasizing parallel throughput over sequential speed. Incoming data are binned in a standard multi-tau scheme with configurable points-per-bin size and are mapped into a GPU memory pattern to reduce time-expensive memory access. Applications include dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS) experiments. We ran the software on a 64-core graphics pci card in a 3.2 GHz Intel i5 CPU based computer running Linux. FCS measurements were made on Alexa-546 and Texas Red dyes in a standard buffer (PBS). Software correlations were compared to hardware correlator measurements on the same signals. Supported by HHMI and Swarthmore College

Real-time measurement of mental workload: A feasibility study

NASA Technical Reports Server (NTRS)

Kramer, Arthur; Humphrey, Darryl; Sirevaag, Erik; Mecklinger, Axel

1990-01-01

The primary goal of the study was to explore the utility of event-related brain potentials (ERP) as real-time measures of workload. To this end, subjects performed two different tasks both separately and together. One task required that subjects monitor a bank of constantly changing gauges and detect critical deviations. Difficulty was varied by changing the predictability of the gauges. The second task was mental arithmetic. Difficulty was varied by requiring subjects to perform operations on either two or three columns of numbers. Two conditions that could easily be distinguished on the basis of performance measures were selected for the real-time evaluation of ERPs. A bootstrapping approach was adopted in which one thousand samples of n trials (n = 1, 3, 5 ...65) were classified using several measures of P300 and Slow Wave amplitude. Classification accuracies of 85 percent were achieved with 25 trials. Results are discussed in terms of potential enhancements for real-time recording.

A real-time high-throughput fluorescence assay for sphingosine kinases

PubMed Central

Lima, Santiago; Milstien, Sheldon; Spiegel, Sarah

2014-01-01

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC50 values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format. PMID:24792926

Novel PDD-PDT system based on spectrophotometric real-time fluorescence monitoring and MALDI-TOF-MS analysis of tumors

NASA Astrophysics Data System (ADS)

Yoshida, Takato O.; Kohno, Eiji; Dodeller, Marc; Sakurai, Takashi; Yamamoto, Seiji; Terakawa, Susumu

2009-06-01

In the PDT practice for tumor patients, the dose and irradiation time for the treatment are chosen by experience and not by real need. To establish advanced PDD-PDT model system for patients, we developed a method for monitoring the cell-death based on a spectrophotometric real-time change in fluorescence in HeLa-tumors during Photofrin®-PDT and ALA-PDT. Here, we describe the results of application of the new PDD-PDT system to human tumors. The fluorescence spectra obtained from human tumors were analyzed by the differential spectral analysis. The mass-spectral changes of tumor tissues during PDD-PDT were also examined by MALDI-TOF-MS/MS. The first author's seborrheic keratosis was monitored with this system during the PDD-PDT with a topically applied ALA-ointment. The changes in fluorescence spectrum were successfully detected, and the tumor regressed completely within 5 months. The differential spectral analysis of PDD-PDT-fluorescence monitoring spectra of tumors and isolated mitochondria showed a marked decrease of three peaks in the red region indicative of the PDD (600 - 720 nm), and a transient rise followed by a decline of peaks in the green region indicative of the PDT (450 - 580 nm). The MALDI-TOF-MS analysis of PDD-PDT HeLa-tumors showed a consumption of Photofrin-deuteroporphyrin and ALA-PpIX, and decreases in protein mass in the range of 4,000 - 16,000 Da, m/z 4929, 8564, 10089, 15000, and an increase in m/z 7002 in a Photofrin® PDD-PDT monitoring tumor.

Real-time detection of dental calculus by blue-LED-induced fluorescence spectroscopy.

PubMed

Qin, Y L; Luan, X L; Bi, L J; Lü, Z; Sheng, Y Q; Somesfalean, G; Zhou, C N; Zhang, Z G

2007-05-25

Successful periodontal therapy requires sensitive techniques to discriminate dental calculus from healthy teeth. The aim of the present study was to develop a fluorescence-based procedure to enable real-time detection and quantification of dental calculus. Thirty human teeth--15 teeth with sub- and supragingival calculus and 15 healthy teeth--covered with a layer of physiological saline solution or blood were illuminated by a focused blue LED light source of 405 nm. Autofluorescence spectra recorded along a randomly selected line stretching over the crown-neck-root area of each tooth were utilized to evaluate a so called calculus parameter R, which was selected to define a relationship between the integrated intensities specific for healthy teeth and for calculus in the 477-497 nm (S(A)) and 628-685 nm (S(B)) wavelength regions, respectively. Statistical analysis was performed and a cut-off threshold of R=0.2 was found to distinguish dental calculus from healthy teeth with 100% sensitivity and specificity under various experimental conditions. The results of the spectral evaluation were confirmed by clinical and histological findings. Automated real-time detection and diagnostics for clinical use were implemented by a corresponding software program written in Visual Basic language. The method enables cost-effective and reliable calculus detection, and can be further developed for imaging applications.

Real-Time Intraoperative Detection of Breast Cancer using Near-infrared Fluorescence Imaging and Methylene Blue

PubMed Central

Tummers, Quirijn R.J.G.; Verbeek, Floris P.R.; Schaafsma, Boudewijn E.; Boonstra, Martin C.; van der Vorst, Joost R.; Liefers, Gerrit-Jan; van de Velde, Cornelis J.H.; Frangioni, John V.; Vahrmeijer, Alexander L.

2014-01-01

Background Despite recent developments in preoperative breast cancer imaging, intraoperative localization of tumor tissue can be challenging, resulting in tumor-positive resection margins during breast-conserving surgery. Based on certain physicochemical similarities between Technetium(99mTc)-sestamibi (MIBI), a SPECT radiodiagnostic with a sensitivity of 83–90% to detect breast cancer preoperatively, and the near-infrared (NIR) fluorophore Methylene Blue (MB), we hypothesized that MB might detect breast cancer intraoperatively using NIR fluorescence imaging. Methods Twenty-four patients with breast cancer, planned for surgical resection, were included. Patients were divided in 2 administration groups, which differed with respect to the timing of MB administration. N = 12 patients per group were administered 1.0 mg/kg MB intravenously either immediately or 3 h before surgery. The mini-FLARE imaging system was used to identify the NIR fluorescent signal during surgery and on post-resected specimens transferred to the pathology department. Results were confirmed by NIR fluorescence microscopy. Results 20/24 (83%) of breast tumors (carcinoma in N=21 and ductal carcinoma in situ in N=3) were identified in the resected specimen using NIR fluorescence imaging. Patients with non-detectable tumors were significantly older. No significant relation to receptor status or tumor grade was seen. Overall tumor-to-background ratio (TBR) was 2.4 ± 0.8. There was no significant difference between TBR and background signal between administration groups. In 2/4 patients with positive resection margins, breast cancer tissue identified in the wound bed during surgery would have changed surgical management. Histology confirmed the concordance of fluorescence signal and tumor tissue. Conclusions This feasibility study demonstrated an overall breast cancer identification rate using MB of 83%, with real-time intraoperative guidance having the potential to alter patient management. PMID

Real-time intraoperative detection of breast cancer using near-infrared fluorescence imaging and Methylene Blue.

PubMed

Tummers, Q R J G; Verbeek, F P R; Schaafsma, B E; Boonstra, M C; van der Vorst, J R; Liefers, G-J; van de Velde, C J H; Frangioni, J V; Vahrmeijer, A L

2014-07-01

Despite recent developments in preoperative breast cancer imaging, intraoperative localization of tumor tissue can be challenging, resulting in tumor-positive resection margins during breast conserving surgery. Based on certain physicochemical similarities between Technetium((99m)Tc)-sestamibi (MIBI), an SPECT radiodiagnostic with a sensitivity of 83-90% to detect breast cancer preoperatively, and the near-infrared (NIR) fluorophore Methylene Blue (MB), we hypothesized that MB might detect breast cancer intraoperatively using NIR fluorescence imaging. Twenty-four patients with breast cancer, planned for surgical resection, were included. Patients were divided in 2 administration groups, which differed with respect to the timing of MB administration. N = 12 patients per group were administered 1.0 mg/kg MB intravenously either immediately or 3 h before surgery. The mini-FLARE imaging system was used to identify the NIR fluorescent signal during surgery and on post-resected specimens transferred to the pathology department. Results were confirmed by NIR fluorescence microscopy. 20/24 (83%) of breast tumors (carcinoma in N = 21 and ductal carcinoma in situ in N = 3) were identified in the resected specimen using NIR fluorescence imaging. Patients with non-detectable tumors were significantly older. No significant relation to receptor status or tumor grade was seen. Overall tumor-to-background ratio (TBR) was 2.4 ± 0.8. There was no significant difference between TBR and background signal between administration groups. In 2/4 patients with positive resection margins, breast cancer tissue identified in the wound bed during surgery would have changed surgical management. Histology confirmed the concordance of fluorescence signal and tumor tissue. This feasibility study demonstrated an overall breast cancer identification rate using MB of 83%, with real-time intraoperative guidance having the potential to alter patient management. Copyright © 2014 Elsevier Ltd. All

A reversible fluorescent probe for real-time live-cell imaging and quantification of endogenous hydropolysulfides.

PubMed

Umezawa, Keitaro; Kamiya, Mako; Urano, Yasuteru

2018-05-23

The chemical biology of reactive sulfur species, including hydropolysulfides, has been a subject undergoing intense study in recent years, but further understanding of their 'intact' function in living cells has been limited due to a lack of appropriate analytical tools. In order to overcome this limitation, we developed a new type of fluorescent probe which reversibly and selectively reacts to hydropolysulfides. The probe enables live-cell visualization and quantification of endogenous hydropolysulfides without interference from intrinsic thiol species such as glutathione. Additionally, real-time reversible monitoring of oxidative-stress-induced fluctuation of intrinsic hydropolysulfides has been achieved with a temporal resolution in the order of seconds, a result which has not yet been realized using conventional methods. These results reveal the probe's versatility as a new fluorescence imaging tool to understand the function of intracellular hydropolysulfides. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A viscosity sensitive fluorescent dye for real-time monitoring of mitochondria transport in neurons.

PubMed

Baek, Yeonju; Park, Sang Jun; Zhou, Xin; Kim, Gyungmi; Kim, Hwan Myung; Yoon, Juyoung

2016-12-15

We present here a viscosity sensitive fluorescent dye, namely thiophene dihemicyanine (TDHC), that enables the specific staining of mitochondria. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this dye demonstrated its unique ability for robust staining of mitochondria with high photostability and ultrahigh signal-to-noise ratio (SNR). Moreover, TDHC also showed high sensitivity towards mitochondria membrane potential (ΔΨm) and intramitochondria viscosity change. Consequently, this dye was utilized in real-time monitoring of mitochondria transport in primary cortical neurons. Finally, the Two-Photon Microscopy (TPM) imaging ability of TDHC was also demonstrated. Copyright © 2016 Elsevier B.V. All rights reserved.

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation.

PubMed

Lee, Jacqueline A; Collings, David A; Glover, Chris N

2016-08-15

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na(+)]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na(+)]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na(+)]i, whereas no significant differences in branchial [Na(+)]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na(+)]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Online fluorescence spectroscopy for the real-time evaluation of the microbial quality of drinking water.

PubMed

Sorensen, J P R; Vivanco, A; Ascott, M J; Gooddy, D C; Lapworth, D J; Read, D S; Rushworth, C M; Bucknall, J; Herbert, K; Karapanos, I; Gumm, L P; Taylor, R G

2018-06-15

We assessed the utility of online fluorescence spectroscopy for the real-time evaluation of the microbial quality of untreated drinking water. Online fluorimeters were installed on the raw water intake at four groundwater-derived UK public water supplies alongside existing turbidity sensors that are used to forewarn of the presence of microbial contamination in the water industry. The fluorimeters targeted fluorescent dissolved organic matter (DOM) peaks at excitation/emission wavelengths of 280/365 nm (tryptophan-like fluorescence, TLF) and 280/450 nm (humic-like fluorescence, HLF). Discrete samples were collected for Escherichia coli, total bacterial cell counts by flow cytometry, and laboratory-based fluorescence and absorbance. Both TLF and HLF were strongly correlated with E. coli (ρ = 0.71-0.77) and total bacterial cell concentrations (ρ = 0.73-0.76), whereas the correlations between turbidity and E. coli (ρ = 0.48) and total bacterial cell counts (ρ = 0.40) were much weaker. No clear TLF peak was observed at the sites and all apparent TLF was considered to be optical bleed-through from the neighbouring HLF peak. Therefore, a HLF fluorimeter alone would be sufficient to evaluate the microbial water quality at these sources. Fluorescent DOM was also influenced by site operations such as pump start-up and the precipitation of cations on the sensor windows. Online fluorescent DOM sensors are a better indicator of the microbial quality of untreated drinking water than turbidity and they have wide-ranging potential applications within the water industry. Copyright © 2018 British Geological Survey, a component institute of NERC - 'BGS © NERC 2018'. Published by Elsevier Ltd.. All rights reserved.

Red-Emitting Fluorescent Probe for Detection of γ-Glutamyltranspeptidase and Its Application of Real-Time Imaging under Oxidative Stress in Cells and in Vivo.

PubMed

Liu, Feiyan; Wang, Zhen; Wang, Wenli; Luo, Jian-Guang; Kong, Lingyi

2018-06-19

γ-Glutamyltranspeptidase (GGT) plays critical roles in regulating various physiological/pathophysiological processes including the intracellular redox homeostasis. However, an effective fluorescent probe for dissecting the relationships between GGT and oxidative stress in vivo remains largely unexplored. Herein, we present a light-up fluorescent probe (DCDHF-Glu) with long wavelength emission (613 nm) for the highly sensitive and selective detection of GGT using dicyanomethylenedihydrofuran derivative as the fluorescent reporter and γ-glutamyl group as the enzyme-active trigger. DCDHF-Glu is competent to real-time image endogenous GGT in live cells and mice. In particular, DCDHF-Glu enables the direct real-time visualization of the upregulation of GGT under drug-induced oxidative stress in the HepG2 cells and the LO2 cells, as well as in vivo, vividly implying its excellent capacity in elucidation of GGT function in GGT-related biological events.

Nondestructive and Real-time Measurement of Moisture in Plant

NASA Astrophysics Data System (ADS)

Ogawa, Yuichi; Kawase, Kodo; Mizuno, Maya; Yamashita, Masatsugu; Otani, Chiko

We constructed a THz transillumination system for water content monitoring, and we succeeded in measuring the moisture level in plants. Our measurement system uses a widely tunable coherent THz parametric oscillator source. As target we chose for this experiment a leaf of Japanese basil. The time variation of the water content in the leaf was monitored in two situations: a leaf freshly cut which is left to dry out, and the leaf of a water stressed plant. We found by real-time measurements that the water content of a cut leaf does not decrease uniformly in time. Also, the response to water stress is delayed by about 5-10 minutes. Furthermore, we demonstrated a moisture measurement using a transillumination THz imaging system. As target we chose for this experiment a leaf of Hedera helix held between two thin plastic sheets. The change of the moisture distribution is clearly visible. These results show that the method described here can be applied to nondestructive and real-time monitoring of water content in plants.

Red fluorescent probes for real-time imaging of the cell cycle by dynamic monitoring of the nucleolus and chromosome.

PubMed

Wang, Kang-Nan; Chao, Xi-Juan; Liu, Bing; Zhou, Dan-Jie; He, Liang; Zheng, Xiao-Hui; Cao, Qian; Tan, Cai-Ping; Zhang, Chen; Mao, Zong-Wan

2018-03-08

Two cationic molecular rotors, 1 and 2, capable of real-time cell-cycle imaging by specifically dynamic monitoring of nucleolus and chromosome changes were developed. A further study shows that fluorescence enhancements in the nucleolus and chromosome are attributed to a combination effect of interaction with nucleic acid and high condensation of the nucleolus and chromosome.

Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

PubMed

Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

2015-03-03

A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

PubMed Central

Vincent, Thomas R.; Canham, James; Toyota, Masatsugu; Avramova, Marieta; Mugford, Sam T.; Gilroy, Simon; Miller, Anthony J.; Hogenhout, Saskia; Sanders, Dale

2017-01-01

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon. PMID:28829425

Reducing background contributions in fluorescence fluctuation time-traces for single-molecule measurements in solution.

PubMed

Földes-Papp, Zeno; Liao, Shih-Chu Jeff; You, Tiefeng; Barbieri, Beniamino

2009-08-01

We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.

A Measure of Real-Time Intelligence

NASA Astrophysics Data System (ADS)

Gavane, Vaibhav

2013-03-01

We propose a new measure of intelligence for general reinforcement learning agents, based on the notion that an agent's environment can change at any step of execution of the agent. That is, an agent is considered to be interacting with its environment in real-time. In this sense, the resulting intelligence measure is more general than the universal intelligence measure (Legg and Hutter, 2007) and the anytime universal intelligence test (Hernández-Orallo and Dowe, 2010). A major advantage of the measure is that an agent's computational complexity is factored into the measure in a natural manner. We show that there exist agents with intelligence arbitrarily close to the theoretical maximum, and that the intelligence of agents depends on their parallel processing capability. We thus believe that the measure can provide a better evaluation of agents and guidance for building practical agents with high intelligence.

Real-time MSE measurements for current profile control on KSTAR.

PubMed

De Bock, M F M; Aussems, D; Huijgen, R; Scheffer, M; Chung, J

2012-10-01

To step up from current day fusion experiments to power producing fusion reactors, it is necessary to control long pulse, burning plasmas. Stability and confinement properties of tokamak fusion reactors are determined by the current or q profile. In order to control the q profile, it is necessary to measure it in real-time. A real-time motional Stark effect diagnostic is being developed at Korean Superconducting Tokamak for Advanced Research for this purpose. This paper focuses on 3 topics important for real-time measurements: minimize the use of ad hoc parameters, minimize external influences and a robust and fast analysis algorithm. Specifically, we have looked into extracting the retardance of the photo-elastic modulators from the signal itself, minimizing the influence of overlapping beam spectra by optimizing the optical filter design and a multi-channel, multiharmonic phase locking algorithm.

Real time in vivo imaging and measurement of serine protease activity in the mouse hippocampus using a dedicated complementary metal-oxide semiconductor imaging device.

PubMed

Ng, David C; Tamura, Hideki; Tokuda, Takashi; Yamamoto, Akio; Matsuo, Masamichi; Nunoshita, Masahiro; Ishikawa, Yasuyuki; Shiosaka, Sadao; Ohta, Jun

2006-09-30

The aim of the present study is to demonstrate the application of complementary metal-oxide semiconductor (CMOS) imaging technology for studying the mouse brain. By using a dedicated CMOS image sensor, we have successfully imaged and measured brain serine protease activity in vivo, in real-time, and for an extended period of time. We have developed a biofluorescence imaging device by packaging the CMOS image sensor which enabled on-chip imaging configuration. In this configuration, no optics are required whereby an excitation filter is applied onto the sensor to replace the filter cube block found in conventional fluorescence microscopes. The fully packaged device measures 350 microm thick x 2.7 mm wide, consists of an array of 176 x 144 pixels, and is small enough for measurement inside a single hemisphere of the mouse brain, while still providing sufficient imaging resolution. In the experiment, intraperitoneally injected kainic acid induced upregulation of serine protease activity in the brain. These events were captured in real time by imaging and measuring the fluorescence from a fluorogenic substrate that detected this activity. The entire device, which weighs less than 1% of the body weight of the mouse, holds promise for studying freely moving animals.

Real-Time Characterization of Virulence Factor Expression in Yersinia pestis Using a Green Fluorescent Protein Reporter System

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

Forde, C; Rocco, J; Fitch, J P

2004-06-09

A real-time reporter system was developed to monitor the thermal induction of virulence factors in Yersinia pestis. The reporter system consists of a plasmid in Y. pestis in which the expression of green fluorescent protein (GFP) is under the control of the promoters for six virulence factors, yopE, sycE, yopK, yopT, yscN, and lcrE/yopN, which are all components of the Type III secretion virulence mechanism of Y. pestis. Induction of the expression of these genes in vivo was determined by the increase in fluorescence intensity of GFP in real time. Basal expression levels observed for the Y. pestis promoters, expressedmore » as percentages of the positive control with GFP under the control of the lac promoter, were: yopE (15%), sycE (15%), yopK (13%), yopT (4%), lcrE (3.3%) and yscN (0.8%). The yopE reporter showed the strongest gene induction following temperature transition from 26 C to 37 C. The induction levels of the other virulence factors, expressed as percentages of yopE induction, were: yopK (57%), sycE (9%), yscN (3%), lcrE (3%), and yopT (2%). The thermal induction of each of these promoter fusions was repressed by calcium, and the ratios of the initial rates of thermal induction without calcium supplementation compared to the rate with calcium supplementation were: yopE (11 fold), yscN (7 fold), yopK (6 fold), lcrE (3 fold), yopT (2 fold), and sycE (2 fold). This work demonstrates a novel approach to quantify gene induction and provides a method to rapidly determine the effects of external stimuli on expression of Y. pestis virulence factors in real time, in living cells.« less

Time-resolved and temperature tuneable measurements of fluorescent intensity using a smartphone fluorimeter.

PubMed

Hossain, Md Arafat; Canning, John; Yu, Zhikang; Ast, Sandra; Rutledge, Peter J; Wong, Joseph K-H; Jamalipour, Abbas; Crossley, Maxwell J

2017-05-30

A smartphone fluorimeter capable of time-based fluorescence intensity measurements at various temperatures is reported. Excitation is provided by an integrated UV LED (λ ex = 370 nm) and detection obtained using the in-built CMOS camera. A Peltier is integrated to allow measurements of the intensity over T = 10 to 40 °C. All components are controlled using a smartphone battery powered Arduino microcontroller and a customised Android application that allows sequential fluorescence imaging and quantification every δt = 4 seconds. The temperature dependence of fluorescence intensity for four emitters (rhodamine B, rhodamine 6G, 5,10,15,20-tetraphenylporphyrin and 6-(1,4,8,11-tetraazacyclotetradecane)2-ethyl-naphthalimide) are characterised. The normalised fluorescence intensity over time of the latter chemosensor dye complex in the presence of Zn 2+ is observed to accelerate with an increasing rate constant, k = 1.94 min -1 at T = 15 °C and k = 3.64 min -1 at T = 30 °C, approaching a factor of ∼2 with only a change in temperature of ΔT = 15 °C. Thermally tuning these twist and bend associated rates to optimise sensor approaches and device applications is proposed.

Real-time structured light intraoral 3D measurement pipeline

NASA Astrophysics Data System (ADS)

Gheorghe, Radu; Tchouprakov, Andrei; Sokolov, Roman

2013-02-01

Computer aided design and manufacturing (CAD/CAM) is increasingly becoming a standard feature and service provided to patients in dentist offices and denture manufacturing laboratories. Although the quality of the tools and data has slowly improved in the last years, due to various surface measurement challenges, practical, accurate, invivo, real-time 3D high quality data acquisition and processing still needs improving. Advances in GPU computational power have allowed for achieving near real-time 3D intraoral in-vivo scanning of patient's teeth. We explore in this paper, from a real-time perspective, a hardware-software-GPU solution that addresses all the requirements mentioned before. Moreover we exemplify and quantify the hard and soft deadlines required by such a system and illustrate how they are supported in our implementation.

In-situ tryptophan-like fluorescence: A real-time indicator of faecal contamination in drinking water supplies.

PubMed

Sorensen, J P R; Lapworth, D J; Marchant, B P; Nkhuwa, D C W; Pedley, S; Stuart, M E; Bell, R A; Chirwa, M; Kabika, J; Liemisa, M; Chibesa, M

2015-09-15

Enteric pathogens are typically inferred from the presence of surrogate indicator organisms such as thermotolerant (faecal) coliforms (TTCs). The analysis of TTCs requires time-consuming incubation in suitable laboratories, which can limit sampling resolution, particularly during critical pollution events. Here, we demonstrate the use of in-situ fluorimeters targeting tryptophan-like compounds as a rapid, reagentless indicator of TTCs in groundwater-derived potable water supplies in Africa. A range of other common indicators of TTCs were also determined including nitrate, turbidity, and sanitary risk survey scores. Sampling was conducted during both the dry and wet seasons to investigate seasonality. Tryptophan-like fluorescence was the most effective predictor of both presence/absence and number of TTCs during both seasons. Seasonal changes in tryptophan-like fluorescence in deeper supplies suggest it is transported more efficiently through the aquifer than TTCs. Moreover, the perennial elevated concentrations in some wells suggest it is more resilient than TTCs in groundwater. Therefore tryptophan-like fluorescence could also be a better indicator of some smaller, more easily transported, and long-lived, pathogenic enteric viruses. These sensors have the potential to be included in real-time pollution alert systems for drinking water supplies throughout the world, as well as for mapping enteric pathogen risks in developing regions. Copyright © 2015 British Geological Survey (a component body of NERC). Published by Elsevier Ltd.. All rights reserved.

Continuous real-time measurement of aqueous cyanide

DOEpatents

Rosentreter, Jeffrey J.; Gering, Kevin L.

2007-03-06

This invention provides a method and system capable of the continuous, real-time measurement of low concentrations of aqueous free cyanide (CN) using an on-line, flow through system. The system is based on the selective reactivity of cyanide anions and the characteristically nonreactive nature of metallic gold films, wherein this selective reactivity is exploited as an indirect measurement for aqueous cyanide. In the present invention the dissolution of gold, due to the solubilization reaction with the analyte cyanide anion, is monitored using a piezoelectric microbalance contained within a flow cell.

Real-time measurements to characterize dynamics of emulsion interface during simulated intestinal digestion.

PubMed

Pan, Yuanjie; Nitin, N

2016-05-01

Efficient delivery of bioactives remains a critical challenge due to their limited bioavailability and solubility. While many encapsulation systems are designed to modulate the digestion and release of bioactives within the human gastrointestinal tract, there is limited understanding of how engineered structures influence the delivery of bioactives. The objective of this study was to develop a real-time quantitative method to measure structural changes in emulsion interface during simulated intestinal digestion and to correlate these changes with the release of free fatty acids (FFAs). Fluorescence resonant energy transfer (FRET) was used for rapid in-situ measurement of the structural changes in emulsion interface during simulated intestinal digestion. By using FRET, changes in the intermolecular spacing between the two different fluorescent probes labeled emulsifier were characterized. Changes in FRET measurements were compared with the release of FFAs. The results showed that bile salts and pancreatic lipase interacted immediately with the emulsion droplets and disrupted the emulsion interface as evidenced by reduction in FRET efficacy compared to the control. Similarly, a significant amount of FFAs was released during digestion. Moreover, addition of a second layer of polymers at emulsion interface decreased the extent of interface disruption by bile salts and pancreatic lipase and impacted the amount or rate of FFA release during digestion. These results were consistent with the lower donor/acceptor ratio of the labeled probes from the FRET result. Overall, this study provides a novel approach to analyze the dynamics of emulsion interface during digestion and their relationship with the release of FFAs. Copyright © 2016 Elsevier B.V. All rights reserved.

Optical see-through cancer vision goggles enable direct patient visualization and real-time fluorescence-guided oncologic surgery

PubMed Central

Mondal, Suman B.; Gao, Shengkui; Zhu, Nan; Hebimana-Griffin, LeMoyne; Akers, Walter J.; Liang, Rongguang; Gruev, Viktor; Margenthaler, Julie; Achilefu, Samuel

2017-01-01

Background The inability to directly visualize the patient and surgical site limits the use of current near infrared fluorescence-guided surgery systems for real-time sentinel lymph node biopsy and tumor margin assessment. Methods We evaluated an optical see-through goggle augmented imaging and navigation system (GAINS) for near-infrared fluorescence-guided surgery. Tumor-bearing mice injected with a near infrared cancer-targeting agent underwent fluorescence-guided tumor resection. Female Yorkshire pigs received hind leg intradermal indocyanine green injection and underwent fluorescence-guided popliteal lymph node resection. Four breast cancer patients received 99mTc-sulfur colloid and indocyanine green retroareolarly, before undergoing sentinel lymph node biopsy using radioactive tracking and fluorescence imaging. Three other breast cancer patients received indocyanine green retroareolarly before undergoing standard-of-care partial mastectomy, followed by fluorescence imaging of resected tumor and tumor cavity for margin assessment. Results Using near-infrared fluorescence from the dyes, the optical see-through GAINS accurately identified all mouse tumors, pig lymphatics, and 4 pig popliteal lymph nodes with high signal-to-background ratio. In 4 human breast cancer patients, 11 sentinel lymph nodes were identified with a detection sensitivity of 86.67± 0.27% for radioactive tracking and 100% for GAINS. Tumor margin status was accurately predicted by GAINS in all three patients, including clear margins in patients 1 and 2 and positive margins in patient 3 as confirmed by paraffin embedded section histopathology. Conclusions The optical see-through GAINS prototype enhances near infrared fluorescence-guided surgery for sentinel lymph node biopsy and tumor margin assessment in breast cancer patients without disrupting the surgical workflow in the operating room. PMID:28213790

Optical See-Through Cancer Vision Goggles Enable Direct Patient Visualization and Real-Time Fluorescence-Guided Oncologic Surgery.

PubMed

Mondal, Suman B; Gao, Shengkui; Zhu, Nan; Habimana-Griffin, LeMoyne; Akers, Walter J; Liang, Rongguang; Gruev, Viktor; Margenthaler, Julie; Achilefu, Samuel

2017-07-01

The inability to visualize the patient and surgical site directly, limits the use of current near infrared fluorescence-guided surgery systems for real-time sentinel lymph node biopsy and tumor margin assessment. We evaluated an optical see-through goggle augmented imaging and navigation system (GAINS) for near-infrared, fluorescence-guided surgery. Tumor-bearing mice injected with a near infrared cancer-targeting agent underwent fluorescence-guided, tumor resection. Female Yorkshire pigs received hind leg intradermal indocyanine green injection and underwent fluorescence-guided, popliteal lymph node resection. Four breast cancer patients received 99m Tc-sulfur colloid and indocyanine green retroareolarly before undergoing sentinel lymph node biopsy using radioactive tracking and fluorescence imaging. Three other breast cancer patients received indocyanine green retroareolarly before undergoing standard-of-care partial mastectomy, followed by fluorescence imaging of resected tumor and tumor cavity for margin assessment. Using near-infrared fluorescence from the dyes, the optical see-through GAINS accurately identified all mouse tumors, pig lymphatics, and four pig popliteal lymph nodes with high signal-to-background ratio. In 4 human breast cancer patients, 11 sentinel lymph nodes were identified with a detection sensitivity of 86.67 ± 0.27% for radioactive tracking and 100% for GAINS. Tumor margin status was accurately predicted by GAINS in all three patients, including clear margins in patients 1 and 2 and positive margins in patient 3 as confirmed by paraffin-embedded section histopathology. The optical see-through GAINS prototype enhances near infrared fluorescence-guided surgery for sentinel lymph node biopsy and tumor margin assessment in breast cancer patients without disrupting the surgical workflow in the operating room.

Time-resolved laser-induced fluorescence system

NASA Astrophysics Data System (ADS)

Bautista, F. J.; De la Rosa, J.; Gallegos, F. J.

2006-02-01

Fluorescence methods are being used increasingly in the measurement of species concentrations in gases, liquids and solids. Laser induced fluorescence is spontaneous emission from atoms or molecules that have been excited by laser radiation. Here we present a time resolved fluorescence instrument that consists of a 5 μJ Nitrogen laser (337.1 nm), a sample holder, a quartz optical fiber, a spectrometer, a PMT and a PC that allows the measurement of visible fluorescence spectra (350-750 nm). Time response of the system is approximately 5 ns. The instrument has been used in the measurement of colored bond paper, antifreeze, diesel, cochineal pigment and malignant tissues. The data acquisition was achieved through computer control of a digital oscilloscope (using General Purpose Interface Bus GPIB) and the spectrometer via serial (RS232). The instrument software provides a graphic interface that lets make some data acquisition tasks like finding fluorescence spectra, and fluorescence lifetimes. The software was developed using the Lab-View 6i graphic programming package and can be easily managed in order to add more functions to it.

Near-infrared fluorescence cholangiography with indocyanine green for biliary atresia. Real-time imaging during the Kasai procedure: a pilot study.

PubMed

Hirayama, Yutaka; Iinuma, Yasushi; Yokoyama, Naoyuki; Otani, Tetsuya; Masui, Daisuke; Komatsuzaki, Naoko; Higashidate, Naruki; Tsuruhisa, Shiori; Iida, Hisataka; Nakaya, Kengo; Naito, Shinichi; Nitta, Koju; Yagi, Minoru

2015-12-01

Hepatoportoenterostomy (HPE) with the Kasai procedure is the treatment of choice for biliary atresia (BA) as the initial surgery. However, the appropriate level of dissection level of the fibrous cone (FC) of the porta hepatis (PH) is frequently unclear, and the procedure sometimes results in unsuccessful outcomes. Recently, indocyanine green near-infrared fluorescence imaging (ICG-FCG) has been developed as a form of real-time cholangiography. We applied this technique in five patients with BA to visualize the biliary flow at the PH intraoperatively. ICG was injected intravenously the day before surgery as the liver function test, and the liver was observed with a near-infrared camera system during the operation while the patient's feces was also observed. In all patients, the whole liver fluoresced diffusely with ICG-containing stagnant bile, whereas no extrahepatic structures fluoresced. The findings of the ICG fluorescence pattern of the PH after dissection of the FC were classified into three types: spotty fluorescence, one patient; diffuse weak fluorescence, three patients; and diffuse strong fluorescence, one patient. In all five patients, the feces evacuated after HPE showed distinct fluorescent spots, although that obtained before surgery showed no fluorescence. One patient with diffuse strong fluorescence who did not achieve JF underwent living related liver transplantation six months after the initial HPE procedure. Four patients, including three cases involving diffuse weak fluorescence and one case involving spotty fluorescence showed weak fluorescence compared to that of the surrounding liver surface. We were able to detect the presence of bile excretion at the time of HPE intraoperatively and successfully evaluated the extent of bile excretion using this new technique. Furthermore, the ICG-FCG findings may provide information leading to a new classification and potentially function as an indicator predicting the clinical outcomes after HPE.

Single-cell real-time imaging of transgene expression upon lipofection

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

Fiume, Giuseppe; Di Rienzo, Carmine; NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127, Pisa

2016-05-20

Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of “symmetry” in protein expression between daughter cells as compared to DC-Chol/DOPE. A model ismore » envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents. -- Highlights: •The process of lipofection is followed by quantifying the transgene expression in real time. •The Lipofectamine gold-standard is compared to the promising DC-Chol/DOPE formulation. •We report that only dividing cells are able to produce the fluorescent reporter protein. •The degree of symmetry of protein expression in daughter cells is linked to DNA bioavailability. •A new experimental platform for the quantitative comparison of transfection reagents is proposed.« less

Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell

2006-01-01

A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

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.

Fluorescence spectroscopy approaches for the development of a real-time organophosphate detection system using an enzymatic sensor.

PubMed

Carullo, Paola; Cetrangolo, Giovanni Paolo; Mandrich, Luigi; Manco, Giuseppe; Febbraio, Ferdinando

2015-02-09

Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.

Development of real-time monitors for gaseous formaldehyde. Final report, 1 December 1988-30 September 1989

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

Kelly, T.J.; Barnes, R.H.

1990-11-01

Two new methods for real-time measurement of gaseous formaldehyde have been developed. One is a spectroscopic method based on direct fluorescence detection of gaseous formaldehyde following excitation with UV light. This method has been developed to the prototype stage by modifications of a commercial fluorescence SO2 detector to convert it to formaldehyde detection. The prototype spectroscopic formaldehyde monitor exhibits a detection limit of <30 ppbv, with a time response of about one minute. The second method is based on derivatization of formaldehyde in aqueous solution to form a fluorescent product. The detection of fluorescent product was made more sensitive bymore » using intense 254 nm light from a mercury lamp for excitation, thereby allowing use of a simple and efficient glass coil scrubber for collection of gaseous formaldehyde. The wet chemical formaldehyde monitor incorportating these improvements exhibits a detection limit for gaseous formaldehyde of 0.2 ppbv and for aqueous formaldehyde of 0.2 micromolar with time response of about one minute, following a lag time of 2 minutes. Both instruments were tested in the laboratory with gaseous formaldehyde standards, and the aqueous scrubbing/analysis method was field tested by continuous operation over a 10-day period in which outdoor and indoor air were sampled for alternate half-hour periods. A comparison of real-time (aqueous scrubbing/analysis) and integrated measurements, using dinitrophenylhydrazine (DNPH) impingers, showed close agreement between the real-time and DNPH data, even at concentrations as low as 1 ppbv.« less

Real-Time Fluorescence Detection in Aqueous Systems by Combined and Enhanced Photonic and Surface Effects in Patterned Hollow Sphere Colloidal Photonic Crystals.

PubMed

Zhong, Kuo; Wang, Ling; Li, Jiaqi; Van Cleuvenbergen, Stijn; Bartic, Carmen; Song, Kai; Clays, Koen

2017-05-16

Hollow sphere colloidal photonic crystals (HSCPCs) exhibit the ability to maintain a high refractive index contrast after infiltration of water, leading to extremely high-quality photonic band gap effects, even in an aqueous (physiological) environment. Superhydrophilic pinning centers in a superhydrophobic environment can be used to strongly confine and concentrate water-soluble analytes. We report a strategy to realize real-time ultrasensitive fluorescence detection in patterned HSCPCs based on strongly enhanced fluorescence due to the photonic band-edge effect combined with wettability differentiation in the superhydrophobic/superhydrophilic pattern. The orthogonal nature of the two strategies allows for a multiplicative effect, resulting in an increase of two orders of magnitude in fluorescence.

[Clinical utility of real-time fluorescent PCR for combined detection of anaplastic lymphoma kinase and c-ros oncogene 1 receptor tyrosine kinase in non-small cell lung cancer].

PubMed

Bai, D Y; Zhang, H P; Zhong, S; Suo, W H; Gao, D H; Ding, Y; Tu, J H

2016-12-23

Objective: To investigate the clinical application value of combined detection of ALK fusion gene and c-ros oncogene 1 receptor tyrosine kinase (ROS1) fusion gene in non-small cell lung cancer (NSCLC) using real-time fluorescent PCR. Methods: A kit for combined detection of ALK fusion gene and ROS1 fusion gene based on fluorescent PCR was used to simultaneously detect the two fusion genes in 302 cases of NSCLC specimens. The results were validated through Sanger sequencing. The consistency of the two detection methods was analyzed. Results: All 302 cases of NSCLC specimens were successfully analyzed through fluorescent PCR (302/302). 12 cases (4.0%) were found to contain ALK fusion gene, including 3 cases with ALK-M1, 3 with ALK-M2, 3 with ALK-M3, 1 with ALK-M4, and 2 with ALK-M6 fusion gene.12 cases (4.0%) were found to contain ROS1 fusion gene, including 1 case with ROS1-M7, 8 cases with ROS1-M8, 1 case with ROS1-M12, 1 case with ROS1-M14, and 1 case with double-positive ROS1-M3 and ROS1-M8 fusion genes. The total detection rate of ALK fusion gene and ROS1 fusion gene was 7.9% (24/302) and 278 cases showed to be negative for ALK fusion gene and ROS1 fusion gene. The successful detection rates for Sanger DNA sequencing were also 100%. The positive, negative and total coincidence rates obtained by real-time fluorescent PCR and by Sanger DNA sequencing were all 100%. Conclusions: The results of Sanger DNA sequencing demonstrate that the real-time fluorescent PCR assay is equally effective in detecting ALK and ROS1 fusion genes in NSCLC tissues. Furthermore, real-time fluorescent PCR assay can be used to detect trace ALK and ROS1 fusion gene simultaneously in tiny samples, and can save time and avoid repeated sampling. It is worthy of recommendation as a rapid and reliable detection technique.

Absolute Density Calibration Cell for Laser Induced Fluorescence Erosion Rate Measurements

NASA Technical Reports Server (NTRS)

Domonkos, Matthew T.; Stevens, Richard E.

2001-01-01

Flight qualification of ion thrusters typically requires testing on the order of 10,000 hours. Extensive knowledge of wear mechanisms and rates is necessary to establish design confidence prior to long duration tests. Consequently, real-time erosion rate measurements offer the potential both to reduce development costs and to enhance knowledge of the dependency of component wear on operating conditions. Several previous studies have used laser-induced fluorescence (LIF) to measure real-time, in situ erosion rates of ion thruster accelerator grids. Those studies provided only relative measurements of the erosion rate. In the present investigation, a molybdenum tube was resistively heated such that the evaporation rate yielded densities within the tube on the order of those expected from accelerator grid erosion. This work examines the suitability of the density cell as an absolute calibration source for LIF measurements, and the intrinsic error was evaluated.

Real-time electron density measurements from Cotton-Mouton effect in JET machine

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

Brombin, M.; Electrical Engineering Department, Padova University, via Gradenigo 6-A, 35131 Padova; Boboc, A.

Real-time density profile measurements are essential for advanced fusion tokamak operation and interferometry is a proven method for this task. Nevertheless, as a consequence of edge localized modes, pellet injections, fast density increases, or disruptions, the interferometer is subject to fringe jumps, which produce loss of the signal preventing reliable use of the measured density in a real-time feedback controller. An alternative method to measure the density is polarimetry based on the Cotton-Mouton effect, which is proportional to the line-integrated electron density. A new analysis approach has been implemented and tested to verify the reliability of the Cotton-Mouton measurements formore » a wide range of plasma parameters and to compare the density evaluated from polarimetry with that from interferometry. The density measurements based on polarimetry are going to be integrated in the real-time control system of JET since the difference with the interferometry is within one fringe for more than 90% of the cases.« less

Real-time navigation system for sentinel lymph node biopsy in breast cancer patients using projection mapping with indocyanine green fluorescence.

PubMed

Takada, Masahiro; Takeuchi, Megumi; Suzuki, Eiji; Sato, Fumiaki; Matsumoto, Yoshiaki; Torii, Masae; Kawaguchi-Sakita, Nobuko; Nishino, Hiroto; Seo, Satoru; Hatano, Etsuro; Toi, Masakazu

2018-05-09

Inability to visualize indocyanine green fluorescence images in the surgical field limits the application of current near-infrared fluorescence imaging (NIR) systems for real-time navigation during sentinel lymph node (SLN) biopsy in breast cancer patients. The aim of this study was to evaluate the usefulness of the Medical Imaging Projection System (MIPS), which uses active projection mapping, for SLN biopsy. A total of 56 patients (59 procedures) underwent SLN biopsy using the MIPS between March 2016 and November 2017. After SLN biopsy using the MIPS, residual SLNs were removed using a conventional NIR camera and/or radioisotope method. The primary endpoint of this study was identification rate of SLNs using the MIPS. In all procedures, at least one SLN was detected by the MIPS, giving an SLN identification rate of 100% [95% confidence interval (CI) 94-100%]. SLN biopsy was successfully performed without operating lights in all procedures. In total, 3 positive SLNs were excised using MIPS, but were not included in the additional SLNs excised by other methods. The median number of SLNs excised using the MIPS was 3 (range 1-7). Of procedures performed after preoperative systemic therapy, the median number of SLNs excised using the MIPS was 3 (range 2-6). The MIPS is effective in detecting SLNs in patients with breast cancer, providing continuous and accurate projection of fluorescence signals in the surgical field, without need for operating lights, and could be useful in real-time navigation surgery for SLN biopsy.

Real-time measurement of soil stiffness during static compaction.

DOT National Transportation Integrated Search

2009-01-01

Is continuous sensing of soil properties during static pad foot roller compaction achievable? A new pad-based, rollerintegrated system for real-time measurement of the elastic modulus of fine- and mixed-grain soils is the goal of Development of So...

Real-time detection of faecally contaminated drinking water with tryptophan-like fluorescence: defining threshold values.

PubMed

Sorensen, James P R; Baker, Andy; Cumberland, Susan A; Lapworth, Dan J; MacDonald, Alan M; Pedley, Steve; Taylor, Richard G; Ward, Jade S T

2018-05-01

We assess the use of fluorescent dissolved organic matter at excitation-emission wavelengths of 280nm and 360nm, termed tryptophan-like fluorescence (TLF), as an indicator of faecally contaminated drinking water. A significant logistic regression model was developed using TLF as a predictor of thermotolerant coliforms (TTCs) using data from groundwater- and surface water-derived drinking water sources in India, Malawi, South Africa and Zambia. A TLF threshold of 1.3ppb dissolved tryptophan was selected to classify TTC contamination. Validation of the TLF threshold indicated a false-negative error rate of 15% and a false-positive error rate of 18%. The threshold was unsuccessful at classifying contaminated sources containing <10 TTC cfu per 100mL, which we consider the current limit of detection. If only sources above this limit were classified, the false-negative error rate was very low at 4%. TLF intensity was very strongly correlated with TTC concentration (ρ s =0.80). A higher threshold of 6.9ppb dissolved tryptophan is proposed to indicate heavily contaminated sources (≥100 TTC cfu per 100mL). Current commercially available fluorimeters are easy-to-use, suitable for use online and in remote environments, require neither reagents nor consumables, and crucially provide an instantaneous reading. TLF measurements are not appreciably impaired by common intereferents, such as pH, turbidity and temperature, within typical natural ranges. The technology is a viable option for the real-time screening of faecally contaminated drinking water globally. Copyright © 2017 Natural Environment Research Council (NERC), as represented by the British Geological Survey (BGS. Published by Elsevier B.V. All rights reserved.

Real-time fluorescence loop-mediated isothermal amplification assay for direct detection of egg drop syndrome virus.

PubMed

Zheney, Makay; Kaziyev, Zhambul; Kassenova, Gulmira; Zhao, Lingna; Liu, Wei; Liang, Lin; Li, Gang

2018-02-13

Egg drop syndrome (EDS), caused by the adenovirus "egg drop syndrome virus" (EDSV) causes severe economic losses through reduced egg production in breeder and layer flocks. The diagnosis of EDSV has been done by molecular tools since its complete genome sequence was identified. In order to enhance the capabilities of the real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay, we aimed to apply the method for direct detection of the EDSV without viral DNA extraction. In order to detect the presence of the EDSV DNA, three pairs of primers were designed, from the conserved region of fiber gene of the EDSV. For our assay, test and control samples were directly used in the reaction mixture in 10-fold serial dilution. The target DNA was amplified at 65 °C, which yield positive results in a relatively short period of 40-45 min. The method reported in this study is highly sensitive as compared to polymerase chain reaction (PCR) and showed no sign of cross-reactivity or false positive results. The RealAmp accomplished specific identification of EDSV among a variety of poultry disease viruses. The direct RealAmp can be used to detect the presence of EDSV. As our result showed, the RealAmp method could be suitable for the direct detection of other DNA viruses.

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells

PubMed Central

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W.; Gautier, Virginie W.

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip. PMID:26485569

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells.

PubMed

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip.

Real-time PCR in virology.

PubMed

Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

2002-03-15

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

Sizing protein-templated gold nanoclusters by time resolved fluorescence anisotropy decay measurements

NASA Astrophysics Data System (ADS)

Soleilhac, Antonin; Bertorelle, Franck; Antoine, Rodolphe

2018-03-01

Protein-templated gold nanoclusters (AuNCs) are very attractive due to their unique fluorescence properties. A major problem however may arise due to protein structure changes upon the nucleation of an AuNC within the protein for any future use as in vivo probes, for instance. In this work, we propose a simple and reliable fluorescence based technique measuring the hydrodynamic size of protein-templated gold nanoclusters. This technique uses the relation between the time resolved fluorescence anisotropy decay and the hydrodynamic volume, through the rotational correlation time. We determine the molecular size of protein-directed AuNCs, with protein templates of increasing sizes, e.g. insulin, lysozyme, and bovine serum albumin (BSA). The comparison of sizes obtained by other techniques (e.g. dynamic light scattering and small-angle X-ray scattering) between bare and gold clusters containing proteins allows us to address the volume changes induced either by conformational changes (for BSA) or the formation of protein dimers (for insulin and lysozyme) during cluster formation and incorporation.

Sizing protein-templated gold nanoclusters by time resolved fluorescence anisotropy decay measurements.

PubMed

Soleilhac, Antonin; Bertorelle, Franck; Antoine, Rodolphe

2018-03-15

Protein-templated gold nanoclusters (AuNCs) are very attractive due to their unique fluorescence properties. A major problem however may arise due to protein structure changes upon the nucleation of an AuNC within the protein for any future use as in vivo probes, for instance. In this work, we propose a simple and reliable fluorescence based technique measuring the hydrodynamic size of protein-templated gold nanoclusters. This technique uses the relation between the time resolved fluorescence anisotropy decay and the hydrodynamic volume, through the rotational correlation time. We determine the molecular size of protein-directed AuNCs, with protein templates of increasing sizes, e.g. insulin, lysozyme, and bovine serum albumin (BSA). The comparison of sizes obtained by other techniques (e.g. dynamic light scattering and small-angle X-ray scattering) between bare and gold clusters containing proteins allows us to address the volume changes induced either by conformational changes (for BSA) or the formation of protein dimers (for insulin and lysozyme) during cluster formation and incorporation. Copyright © 2017 Elsevier B.V. All rights reserved.

Dual turn-on fluorescence signal-based controlled release system for real-time monitoring of drug release dynamics in living cells and tumor tissues.

PubMed

Kong, Xiuqi; Dong, Baoli; Song, Xuezhen; Wang, Chao; Zhang, Nan; Lin, Weiying

2018-01-01

Controlled release systems with capabilities for direct and real-time monitoring of the release and dynamics of drugs in living systems are of great value for cancer chemotherapy. Herein, we describe a novel dual turn-on fluorescence signal-based controlled release system ( CDox ), in which the chemotherapy drug doxorubicin ( Dox ) and the fluorescent dye ( CH ) are conjugated by a hydrazone moiety, a pH-responsive cleavable linker. CDox itself shows nearly no fluorescence as the fluorescence of CH and Dox is essentially quenched by the C=N isomerization and N-N free rotation. However, when activated under acidic conditions, CDox could be hydrolyzed to afford Dox and CH , resulting in dual turn-on signals with emission peaks at 595 nm and 488 nm, respectively. Notably, CDox exhibits a desirable controlled release feature as the hydrolysis rate is limited by the steric hindrance effect from both the Dox and CH moieties. Cytotoxicity assays indicate that CDox shows much lower cytotoxicity relative to Dox , and displays higher cell inhibition rate to cancer than normal cells. With the aid of the dual turn-on fluorescence at different wavelengths, the drug release dynamics of CDox in living HepG2 and 4T-1 cells was monitored in double channels in a real-time fashion. Importantly, two-photon fluorescence imaging of CDox in living tumor tissues was also successfully performed by high-definition 3D imaging. We expect that the unique controlled release system illustrated herein could provide a powerful means to investigate modes of action of drugs, which is critical for development of much more robust and effective chemotherapy drugs.

A new front-face optical cell for measuring weak fluorescent emissions with time resolution in the picosecond time scale.

PubMed

Gryczynski, Z; Bucci, E

1993-11-01

Recent developments of ultrafast fluorimeters allow measuring time-resolved fluorescence on the picosecond time scale. This implies one is able to monitor lifetimes and anisotropy decays of highly quenched systems and of systems that contain fluorophores having lifetimes in the subnanosecond range; both systems that emit weak signals. The combination of weak signals and very short lifetimes makes the measurements prone to distortions which are negligible in standard fluorescence experiments. To cope with these difficulties, we have designed a new optical cell for front-face optics which offers to the excitation beam a horizontal free liquid surface in the absence of interactions with optical windows. The new cell has been tested with probes of known lifetimes and anisotropies. It proved very useful in detecting tryptophan fluorescence in hemoglobin. If only diluted samples are available, which cannot be used in front-face optics, regular square geometry can still be utilized by inserting light absorbers into a cuvette of 1 cm path length.

Ruggedized downhole tool for real-time measurements and uses thereof

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

Hess, Ryan Falcone; Lindblom, Scott C.; Yelton, William G.

The present invention relates to ruggedized downhole tools and sensors, as well as uses thereof. In particular, these tools can operate under extreme conditions and, therefore, allow for real-time measurements in geothermal reservoirs or other potentially harsh environments. One exemplary sensor includes a ruggedized ion selective electrode (ISE) for detecting tracer concentrations in real-time. In one embodiment, the ISE includes a solid, non-conductive potting material and an ion selective material, which are disposed in a temperature-resistant electrode body. Other electrode configurations, tools, and methods are also described.

Flexible imaging payload for real-time fluorescent biological imaging in parabolic, suborbital and space analog environments

NASA Astrophysics Data System (ADS)

Bamsey, Matthew T.; Paul, Anna-Lisa; Graham, Thomas; Ferl, Robert J.

2014-10-01

utilized to assess, in real-time, the response of plants to novel environments including various spaceflight analogs, including several parabolic flight environments as well as hypobaric plant growth chambers. Basic performance results obtained under these operational environments, as well as laboratory-based tests are described. The Flex Imager has also been designed to be compatible with emerging suborbital platforms.

Laparoscopic detection and resection of occult liver tumors of multiple cancer types using real-time near-infrared fluorescence guidance.

PubMed

Boogerd, Leonora S F; Handgraaf, Henricus J M; Lam, Hwai-Ding; Huurman, Volkert A L; Farina-Sarasqueta, Arantza; Frangioni, John V; van de Velde, Cornelis J H; Braat, Andries E; Vahrmeijer, Alexander L

2017-02-01

Tumor recurrence after radical resection of hepatic tumors is not uncommon, suggesting that malignant lesions are missed during surgery. Intraoperative navigation using fluorescence guidance is an innovative technique enabling real-time identification of (sub)capsular liver tumors. The objective of the current study was to compare fluorescence imaging (FI) and conventional imaging modalities for laparoscopic detection of both primary and metastatic tumors in the liver. Patients undergoing laparoscopic resection of a malignant hepatic tumor were eligible for inclusion. Patients received standard of care, including preoperative CT and/or MRI. In addition, 10 mg indocyanine green was intravenously administered 1 day prior to surgery. After introduction of the laparoscope, inspection, FI, and laparoscopic ultrasonography (LUS) were performed. Histopathological examination of resected suspect tissue was considered the gold standard. Twenty-two patients suspected of having hepatocellular carcinoma (n = 4), cholangiocarcinoma (n = 2) or liver metastases from colorectal carcinoma (n = 12), uveal melanoma (n = 2), and breast cancer (n = 2) were included. Two patients were excluded because their surgery was unexpectedly postponed several days. Twenty-six malignancies were resected in the remaining 20 patients. Sensitivity for various modalities was 80 % (CT), 84 % (MRI), 62 % (inspection), 86 % (LUS), and 92 % (FI), respectively. Three metastases (12 %) were identified solely by FI. All 26 malignancies could be detected by combining LUS and FI (100 % sensitivity). This study demonstrates added value of FI during laparoscopic resections of several hepatic tumors. Although larger series will be needed to confirm long-term patient outcome, the technology already aids the surgeon by providing real-time fluorescence guidance.

In vivo and in vitro characterization of σ70 constitutive promoters by real-time PCR and fluorescent measurements.

PubMed

Chappell, James; Freemont, Paul

2013-01-01

The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ(70) transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction.

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers.

PubMed

Lichten, Catherine A; White, Rachel; Clark, Ivan B N; Swain, Peter S

2014-02-03

To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour.

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers

PubMed Central

2014-01-01

Background To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. Results Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). Conclusions Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour. PMID:24495318

FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping.

PubMed

Ansart-Pirenne, H; Martin-Blanc, S; Le Pennec, P-Y; Rouger, P; Cartron, J-P; Tournamille, C

2007-02-01

The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.

Optical mapping system with real-time control capability.

PubMed

Iravanian, Shahriar; Christini, David J

2007-10-01

Real-time, closed-loop intervention is an emerging experiment-control method that promises to provide invaluable new insight into cardiac electrophysiology. One example is the investigation of closed-loop feedback control of cardiac activity (e.g., alternans) as a possible method of preventing arrhythmia onset. To date, such methods have been investigated only in vitro using microelectrode systems, which are hindered by poor spatial resolution and are not well suited for atrial or ventricular tissue preparations. We have developed a system that uses optical mapping techniques and an electrical stimulator as the sensory and effector arms, respectively, of a closed-loop, real-time control system. The system consists of a 2,048 x 1 pixel line-scan charge-coupled device camera that records optical signals from the tissue. Custom-image processing and control software, which is implemented on top of a hard real-time operation system (RTAI Linux), process the data and make control decisions with a deterministic delay of <1 ms. The system is tested in two ways: 1) it is used to control, in real time, simulated optical signals of electrical alternans; and 2) it uses precisely timed, feedback-controlled initiation of antitachycardia pacing to terminate reentrant arrhythmias in an arterially perfused swine right ventricle stained with voltage-sensitive fluorescent dye 4{beta-[2-(di-n-butylamino)-6-napathy]vinyl}pyridinium (di-4-ANEPPS). Thus real-time control of cardiac activity using optical mapping techniques is feasible. Such a system is attractive because it offers greater measurement resolution than the electrode-based systems with which real-time control has been used previously.

Protein Analysis Using Real-Time PCR Instrumentation: Incorporation in an Integrated, Inquiry-Based Project

ERIC Educational Resources Information Center

Southard, Jonathan N.

2014-01-01

Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein…

Real-Time Inhibitor Recession Measurements in the Space Shuttle Reusable Solid Rocket Motors

NASA Technical Reports Server (NTRS)

McWhorter, Bruce B.; Ewing, Mark E.; McCool, Alex (Technical Monitor)

2001-01-01

Real-time char line recession measurements were made on propellant inhibitors of the Space Shuttle Reusable Solid Rocket Motor (RSRM). The RSRM FSM-8 static test motor propellant inhibitors (composed of a rubber insulation material) were successfully instrumented with eroding potentiometers and thermocouples. The data was used to establish inhibitor recession versus time relationships. Normally, pre-fire and post-fire insulation thickness measurements establish the thermal performance of an ablating insulation material. However, post-fire inhibitor decomposition and recession measurements are complicated by the fact that most of the inhibitor is back during motor operation. It is therefore a difficult task to evaluate the thermal protection offered by the inhibitor material. Real-time measurements would help this task. The instrumentation program for this static test motor marks the first time that real-time inhibitors. This report presents that data for the center and aft field joint forward facing inhibitors. The data was primarily used to measure char line recession of the forward face of the inhibitors which provides inhibitor thickness reduction versus time data. The data was also used to estimate the inhibitor height versus time relationship during motor operation.

Using fluorescence lymphangiography to define the ileocolic mesentery: proof of concept for the watershed area using real-time imaging.

PubMed

Keller, D S; Joshi, H M; Rodriguez-Justo, M; Walsh, D; Coffey, J C; Chand, M

2017-09-01

Recent advances in mesenteric science have demonstrated that the mesentery is a continuous structure with a 'watershed' area at the mesenteric apex between the right colon and terminal ileum, where lymphatic flow can proceed either proximally or distally. With this new understanding of the anatomy, functional features are emerging, which can have an impact on surgical management. Fluorescence lymphangiography or lymphoscintigraphy with indocyanine green allows real-time visualization of lymphatic channels, which highlights sentinel lymph nodes and may facilitate identification of the ideal margins for mesenteric lymphadenectomy during bowel resection for colon cancer. By using this novel technology, it is possible to demonstrate a watershed area in the ileocolic region and may facilitate more precise mesenteric dissection. In the present study, we provide proof of concept for the ileocolic watershed area using fluorescence lymphangiography.

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

PubMed

Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

2013-01-01

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

Real-time and high accuracy frequency measurements for intermediate frequency narrowband signals

NASA Astrophysics Data System (ADS)

Tian, Jing; Meng, Xiaofeng; Nie, Jing; Lin, Liwei

2018-01-01

Real-time and accurate measurements of intermediate frequency signals based on microprocessors are difficult due to the computational complexity and limited time constraints. In this paper, a fast and precise methodology based on the sigma-delta modulator is designed and implemented by first generating the twiddle factors using the designed recursive scheme. This scheme requires zero times of multiplications and only half amounts of addition operations by using the discrete Fourier transform (DFT) and the combination of the Rife algorithm and Fourier coefficient interpolation as compared with conventional methods such as DFT and Fast Fourier Transform. Experimentally, when the sampling frequency is 10 MHz, the real-time frequency measurements with intermediate frequency and narrowband signals have a measurement mean squared error of ±2.4 Hz. Furthermore, a single measurement of the whole system only requires approximately 0.3 s to achieve fast iteration, high precision, and less calculation time.

Real-time long term measurement using integrated framework for ubiquitous smart monitoring

NASA Astrophysics Data System (ADS)

Heo, Gwanghee; Lee, Giu; Lee, Woosang; Jeon, Joonryong; Kim, Pil-Joong

2007-04-01

Ubiquitous monitoring combining internet technologies and wireless communication is one of the most promising technologies of infrastructure health monitoring against the natural of man-made hazards. In this paper, an integrated framework of the ubiquitous monitoring is developed for real-time long term measurement in internet environment. This framework develops a wireless sensor system based on Bluetooth technology and sends measured acceleration data to the host computer through TCP/IP protocol. And it is also designed to respond to the request of web user on real time basis. In order to verify this system, real time monitoring tests are carried out on a prototype self-anchored suspension bridge. Also, wireless measurement system is analyzed to estimate its sensing capacity and evaluate its performance for monitoring purpose. Based on the evaluation, this paper proposes the effective strategies for integrated framework in order to detect structural deficiencies and to design an early warning system.

A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

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

Jothikumar, N., E-mail: jin2@cdc.gov; Hill, Vincent R.

Highlights: •Uses a single-labeled fluorescent primer for real-time PCR. •The detection sensitivity of PET PCR was comparable to TaqMan PCR. •Melt curve analysis can be performed to confirm target amplicon production. •Conventional PCR primers can be converted to PET PCR primers. -- Abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forwardmore » or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource

A real-time single sperm tracking, laser trapping, and ratiometric fluorescent imaging system

NASA Astrophysics Data System (ADS)

Shi, Linda Z.; Botvinick, Elliot L.; Nascimento, Jaclyn; Chandsawangbhuwana, Charlie; Berns, Michael W.

2006-08-01

Sperm cells from a domestic dog were treated with oxacarbocyanine DiOC II(3), a ratiometrically-encoded membrane potential fluorescent probe in order to monitor the mitochondria stored in an individual sperm's midpiece. This dye normally emits a red fluorescence near 610 nm as well as a green fluorescence near 515 nm. The ratio of red to green fluorescence provides a substantially accurate and precise measurement of sperm midpiece membrane potential. A two-level computer system has been developed to quantify the motility and energetics of sperm using video rate tracking, automated laser trapping (done by the upper-level system) and fluorescent imaging (done by the lower-level system). The communication between these two systems is achieved by a networked gigabit TCP/IP cat5e crossover connection. This allows for the curvilinear velocity (VCL) and ratio of the red to green fluorescent images of individual sperm to be written to the hard drive at video rates. This two-level automatic system has increased experimental throughput over our previous single-level system (Mei et al., 2005) by an order of magnitude.

Subcellular real-time in vivo imaging of intralymphatic and intravascular cancer-cell trafficking

NASA Astrophysics Data System (ADS)

McElroy, M.; Hayashi, K.; Kaushal, S.; Bouvet, M.; Hoffman, Robert M.

2008-02-01

With the use of fluorescent cells labeled with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and a highly sensitive small animal imaging system with both macro-optics and micro-optics, we have developed subcellular real-time imaging of cancer cell trafficking in live mice. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 small animal imaging system with a sensitive CCD camera and four objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. We have also developed real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with GFP and/or RFP were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real-time at the cellular level until they entered the axillary lymph node. The bright dual-color fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 enabled imaging the trafficking cancer cells in both blood vessels and lymphatics. With the dual-color cancer cells and the highly sensitive imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time.

[Experimental studies of using real-time fluorescence quantitative PCR and RT-PCR to detect E6 and E7 genes of human papillomavirus type 16 in cervical carcinoma cell lines].

PubMed

Chen, Yue-yue; Peng, Zhi-lan; Liu, Shan-ling; He, Bing; Hu, Min

2007-06-01

To establish a method of using real-time fluorescence quantitative PCR and RT-PCR to detect the E6 and E7 genes of human papillomavirus type 16 (HPV-16). Plasmids containing HPV-16 E6 or E7 were used to generate absolute standard curves. Three cervical carcinoma cell lines CaSki, SiHa and HeLa were tested by real-time fluorescence quantitative PCR and RT-PCR analyses for the expressions of HPV-16 E6 and E7. The correlation coefficients of standard curves were larger than 0. 99, and the PCR efficiency was more than 90%. The relative levels of HPV-16 E6 and E7 DNA and RNA were CaSki>SiHa>HeLa cell. HPV-16 E6 and E7 quantum by real-time fluorescence quantitative PCR and RT-PCR analyses may serve as a reliable and sensitive tool. This study provides the possibility of further researches on the relationship between HPV-16 E6 or E7 copy number and cervical carcinoma.

Characterization of real-time computers

NASA Technical Reports Server (NTRS)

Shin, K. G.; Krishna, C. M.

1984-01-01

A real-time system consists of a computer controller and controlled processes. Despite the synergistic relationship between these two components, they have been traditionally designed and analyzed independently of and separately from each other; namely, computer controllers by computer scientists/engineers and controlled processes by control scientists. As a remedy for this problem, in this report real-time computers are characterized by performance measures based on computer controller response time that are: (1) congruent to the real-time applications, (2) able to offer an objective comparison of rival computer systems, and (3) experimentally measurable/determinable. These measures, unlike others, provide the real-time computer controller with a natural link to controlled processes. In order to demonstrate their utility and power, these measures are first determined for example controlled processes on the basis of control performance functionals. They are then used for two important real-time multiprocessor design applications - the number-power tradeoff and fault-masking and synchronization.

Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

NASA Astrophysics Data System (ADS)

Jaiswal, Devina; Rad, Armin Tahmasbi; Nieh, Mu-Ping; Claffey, Kevin P.; Hoshino, Kazunori

2017-04-01

Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 μg/ml and 0.08 μg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.

Real-time fluorescence assay of alkaline phosphatase in living cells using boron-doped graphene quantum dots as fluorophores.

PubMed

Chen, Li; Yang, Guancao; Wu, Ping; Cai, Chenxin

2017-10-15

This work reports a convenient and real-time assay of alkaline phosphatase (ALP) in living cells based on a fluorescence quench-recovery process at a physiological pH using the boron-doped graphene quantum dots (BGQDs) as fluorophore. The fluorescence of BGQDs is found to be effectively quenched by Ce 3+ ions because of the coordination of Ce 3+ ions with the carboxyl group of BGQDs. Upon addition of adenosine triphosphate (ATP) into the system, the quenched fluorescence can be recovered by the ALP-positive expressed cells (such as MCF-7 cells) due to the removal of Ce 3+ ions from BGQDs surface by phosphate ions, which are generated from ATP under catalytic hydrolysis of ALP that expressed in cells. The extent of fluorescence signal recovery depends on the level of ALP in cells, which establishes the basis of ALP assay in living cells. This approach can also be used for specific discrimination of the ALP expression levels in different type of cells and thus sensitive detection of those ALP-positive expressed cells (for example MCF-7 cells) at a very low abundance (10±5 cells mL -1 ). The advantages of this approach are that it has high sensitivity because of the significant suppression of the background due to the Ce 3+ ion quenching the fluorescence of BGQDs, and has the ability of avoiding false signals arising from the nonspecific adsorption of non-target proteins because it operates via a fluorescence quench-recovery process. In addition, it can be extended to other enzyme systems, such as ATP-related kinases. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-time diamagnetic flux measurements on ASDEX Upgrade.

PubMed

Giannone, L; Geiger, B; Bilato, R; Maraschek, M; Odstrčil, T; Fischer, R; Fuchs, J C; McCarthy, P J; Mertens, V; Schuhbeck, K H

2016-05-01

Real-time diamagnetic flux measurements are now available on ASDEX Upgrade. In contrast to the majority of diamagnetic flux measurements on other tokamaks, no analog summation of signals is necessary for measuring the change in toroidal flux or for removing contributions arising from unwanted coupling to the plasma and poloidal field coil currents. To achieve the highest possible sensitivity, the diamagnetic measurement and compensation coil integrators are triggered shortly before plasma initiation when the toroidal field coil current is close to its maximum. In this way, the integration time can be chosen to measure only the small changes in flux due to the presence of plasma. Two identical plasma discharges with positive and negative magnetic field have shown that the alignment error with respect to the plasma current is negligible. The measured diamagnetic flux is compared to that predicted by TRANSP simulations. The poloidal beta inferred from the diamagnetic flux measurement is compared to the values calculated from magnetic equilibrium reconstruction codes. The diamagnetic flux measurement and TRANSP simulation can be used together to estimate the coupled power in discharges with dominant ion cyclotron resonance heating.

Toward real-time quantification of fluorescence molecular probes using target/background ratio for guiding biopsy and endoscopic therapy of esophageal neoplasia.

PubMed

Jiang, Yang; Gong, Yuanzheng; Rubenstein, Joel H; Wang, Thomas D; Seibel, Eric J

2017-04-01

Multimodal endoscopy using fluorescence molecular probes is a promising method of surveying the entire esophagus to detect cancer progression. Using the fluorescence ratio of a target compared to a surrounding background, a quantitative value is diagnostic for progression from Barrett's esophagus to high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). However, current quantification of fluorescent images is done only after the endoscopic procedure. We developed a Chan-Vese-based algorithm to segment fluorescence targets, and subsequent morphological operations to generate background, thus calculating target/background (T/B) ratios, potentially to provide real-time guidance for biopsy and endoscopic therapy. With an initial processing speed of 2 fps and by calculating the T/B ratio for each frame, our method provides quasireal-time quantification of the molecular probe labeling to the endoscopist. Furthermore, an automatic computer-aided diagnosis algorithm can be applied to the recorded endoscopic video, and the overall T/B ratio is calculated for each patient. The receiver operating characteristic curve was employed to determine the threshold for classification of HGD/EAC using leave-one-out cross-validation. With 92% sensitivity and 75% specificity to classify HGD/EAC, our automatic algorithm shows promising results for a surveillance procedure to help manage esophageal cancer and other cancers inspected by endoscopy.

Real-time measurements of airborne biologic particles using fluorescent particle counter to evaluate microbial contamination: results of a comparative study in an operating theater.

PubMed

Dai, Chunyang; Zhang, Yan; Ma, Xiaoling; Yin, Meiling; Zheng, Haiyang; Gu, Xuejun; Xie, Shaoqing; Jia, Hengmin; Zhang, Liang; Zhang, Weijun

2015-01-01

Airborne bacterial contamination poses a risk for surgical site infection, and routine surveillance of airborne bacteria is important. Traditional methods for detecting airborne bacteria are time consuming and strenuous. Measurement of biologic particle concentrations using a fluorescent particle counter is a novel method for evaluating air quality. The current study was to determine whether the number of biologic particles detected by the fluorescent particle counter can be used to indicate airborne bacterial counts in operating rooms. The study was performed in an operating theater at a university hospital in Hefei, China. The number of airborne biologic particles every minute was quantified using a fluorescent particle counter. Microbiologic air sampling was performed every 30 minutes using an Andersen air sampler (Pusong Electronic Instruments, Changzhou, China). Correlations between the 2 different methods were analyzed by Pearson correlation coefficients. A significant correlation was observed between biologic particle and bacterial counts (Pearson correlation coefficient = 0.76), and the counting results from 2 methods both increased substantially between operations, corresponding with human movements in the operating room. Fluorescent particle counters show potential as important tools for monitoring bacterial contamination in operating theatres. Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Real-Time Stability Margin Measurements for X-38 Robustness Analysis

NASA Technical Reports Server (NTRS)

Bosworth, John T.; Stachowiak, Susan J.

2005-01-01

A method has been developed for real-time stability margin measurement calculations. The method relies on a tailored-forced excitation targeted to a specific frequency range. Computation of the frequency response is matched to the specific frequencies contained in the excitation. A recursive Fourier transformation is used to make the method compatible with real-time calculation. The method was incorporated into the X-38 nonlinear simulation and applied to an X-38 robustness test. X-38 stability margins were calculated for different variations in aerodynamic and mass properties over the vehicle flight trajectory. The new method showed results comparable to more traditional stability analysis techniques, and at the same time, this new method provided coverage that is more complete and increased efficiency.

Real-time micro-vibration multi-spot synchronous measurement within a region based on heterodyne interference

NASA Astrophysics Data System (ADS)

Lan, Ma; Xiao, Wen; Chen, Zonghui; Hao, Hongliang; Pan, Feng

2018-01-01

Real-time micro-vibration measurement is widely used in engineering applications. It is very difficult for traditional optical detection methods to achieve real-time need in a relatively high frequency and multi-spot synchronous measurement of a region at the same time,especially at the nanoscale. Based on the method of heterodyne interference, an experimental system of real-time measurement of micro - vibration is constructed to satisfy the demand in engineering applications. The vibration response signal is measured by combing optical heterodyne interferometry and a high-speed CMOS-DVR image acquisition system. Then, by extracting and processing multiple pixels at the same time, four digital demodulation technique are implemented to simultaneously acquire the vibrating velocity of the target from the recorded sequences of images. Different kinds of demodulation algorithms are analyzed and the results show that these four demodulation algorithms are suitable for different interference signals. Both autocorrelation algorithm and cross-correlation algorithm meet the needs of real-time measurements. The autocorrelation algorithm demodulates the frequency more accurately, while the cross-correlation algorithm is more accurate in solving the amplitude.

Breaking Out of the Lab: Measuring Real-Time Responses to Televised Political Content in Real-World Settings.

PubMed

Maier, Jürgen; Hampe, J Felix; Jahn, Nico

2016-01-01

Real-time response (RTR) measurement is an important technique for analyzing human processing of electronic media stimuli. Although it has been demonstrated that RTR data are reliable and internally valid, some argue that they lack external validity. The reason for this is that RTR measurement is restricted to a laboratory environment due to its technical requirements. This paper introduces a smartphone app that 1) captures real-time responses using the dial technique and 2) provides a solution for one of the most important problems in RTR measurement, the (automatic) synchronization of RTR data. In addition, it explores the reliability and validity of mobile RTR measurement by comparing the real-time reactions of two samples of young and well-educated voters to the 2013 German televised debate. Whereas the first sample participated in a classical laboratory study, the second sample was equipped with our mobile RTR system and watched the debate at home. Results indicate that the mobile RTR system yields similar results to the lab-based RTR measurement, providing evidence that laboratory studies using RTR are externally valid. In particular, the argument that the artificial reception situation creates artificial results has to be questioned. In addition, we conclude that RTR measurement outside the lab is possible. Hence, mobile RTR opens the door for large-scale studies to better understand the processing and impact of electronic media content.

A real-time fluorescence assay for protease activity and inhibitor screening based on the aggregation-caused quenching of a perylene probe.

PubMed

Wang, Yan; Zhang, Zhifang; Zhang, Ya; Yu, Cong

2018-06-01

We have established a real-time and label-free fluorescence turn-on strategy for protease activity detection and inhibitor screening via peptide-induced aggregation-caused quenching of a perylene probe. Because of electrostatic interactions and high hydrophilicity, poly-l-glutamic acid sodium salt (PGA; a negatively charged peptide) could induce aggregation of a positively charged perylene probe (probe 1) and the monomer fluorescence of probe 1 was effectively quenched. After a protease was added, PGA was enzymatically hydrolyzed into small fragments and probe 1 disaggregated. The fluorescence recovery of probe 1 was found to be proportional to the concentration of protease in the range from 0 to 1 mU/ml. The detection limit was down to 0.1 mU/ml. In the presence of a protease inhibitor, protease activity was inhibited and fluorescence recovery reduced. Moreover, we demonstrated the potential application of our method in a complex mixture sample including 1% human serum. Our method is simple, fast and cost effective. Copyright © 2018 John Wiley & Sons, Ltd.

Fluorescent Silicon Nanorods-Based Ratiometric Sensors for Long-Term and Real-Time Measurements of Intracellular pH in Live Cells.

PubMed

Chu, Binbin; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; Wang, Houyu; He, Yao

2017-11-21

Long-term and real-time investigation of the dynamic process of pH i changes is critically significant for understanding the related pathogenesis of diseases and the design of intracellular drug delivery systems. Herein, we present a one-step synthetic strategy to construct ratiometric pH sensors, which are made of europium (Eu)-doped one-dimensional silicon nanorods (Eu@SiNRs). The as-prepared Eu@SiNRs have distinct emission maxima peaks at 470 and 620 nm under 405 nm excitation. Of particular note, the fluorescence emission intensity at 470 nm decreases along with the increase of pH, while the one at 620 nm is nearly unaffected by pH changes, making Eu@SiNRs a feasible probe for pH sensing ratiometrically. Moreover, Eu@SiNRs are found to be responsive to a broad pH range (ca. 3-9), biocompatible (e.g., ∼100% of cell viability during 24 h treatment) and photostable (e.g., ∼10% loss of intensity after 40 min continuous UV irradiation). Taking advantages of these merits, we employ Eu@SiNRs for the visualization of the cytoplasmic alkalization process mediated by nigericin in living cells, for around 30 min without interruption, revealing important information for understanding the dynamic process of pH i fluctuations.

Integrated Raman spectroscopy and trimodal wide-field imaging techniques for real-time in vivo tissue Raman measurements at endoscopy.

PubMed

Huang, Zhiwei; Teh, Seng Khoon; Zheng, Wei; Mo, Jianhua; Lin, Kan; Shao, Xiaozhuo; Ho, Khek Yu; Teh, Ming; Yeoh, Khay Guan

2009-03-15

We report an integrated Raman spectroscopy and trimodal (white-light reflectance, autofluorescence, and narrow-band) imaging techniques for real-time in vivo tissue Raman measurements at endoscopy. A special 1.8 mm endoscopic Raman probe with filtering modules is developed, permitting effective elimination of interference of fluorescence background and silica Raman in fibers while maximizing tissue Raman collections. We demonstrate that high-quality in vivo Raman spectra of upper gastrointestinal tract can be acquired within 1 s or subseconds under the guidance of wide-field endoscopic imaging modalities, greatly facilitating the adoption of Raman spectroscopy into clinical research and practice during routine endoscopic inspections.

Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo

PubMed Central

Yang, Jin-Long; Cheng, An-Chun; Wang, Ming-Shu; Pan, Kang-Cheng; Li, Min; Guo, Yu-Fei; Li, Chuan-Feng; Zhu, De-Kang; Chen, Xiao-Yue

2009-01-01

Background Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, it severely affects geese production worldwide. The objective of this study was to develop a fluorescent quantitative real-time polymerase chain reaction (PCR) (FQ-PCR) assay for fast and accurate quantification of GPV DNA in infected goslings, which can aid in the understanding of the regular distribution pattern and the nosogenesis of GPV in vivo. Results The detection limit of the assay was 2.8 × 101 standard DNA copies, with a sensitivity of 3 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intraassay and interassay coefficients of variation. Conclusion The high sensitivity, specificity, simplicity, and reproducibility of the GPV fluorogenic PCR assay, combined with a high throughput, make this method suitable for a broad spectrum of GPV etiology-related applications. PMID:19754946

Rapid detection of Wuchereria bancrofti and Brugia malayi in mosquito vectors (Diptera: Culicidae) using a real-time fluorescence resonance energy transfer multiplex PCR and melting curve analysis.

PubMed

Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Maleewong, Wanchai

2009-01-01

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate Lymphatic Filariasis.

Evaluation of Impermeant, DNA-Binding Dye Fluorescence as a Real-Time Readout of Eukaryotic Cell Toxicity in a High Throughput Screening Format

PubMed Central

Chiaraviglio, Lucius

2014-01-01

Abstract Interpretation of high throughput screening (HTS) data in cell-based assays may be confounded by cytotoxic properties of screening compounds. Therefore, assessing cell toxicity in real time during the HTS process itself would be highly advantageous. Here, we investigate the potential of putatively impermeant, fluorescent, DNA-binding dyes to give cell toxicity readout during HTS. Amongst 19 DNA-binding dyes examined, three classes were identified that were (1) permeant, (2) cytotoxic, or (3) neither permeant nor cytotoxic during 3-day incubation with a macrophage cell line. In the last class, four dyes (SYTOX Green, CellTox Green, GelGreen, and EvaGreen) gave highly robust cytotoxicity data in 384-well screening plates. As proof of principle, successful combination with a luminescence-based assay in HTS format was demonstrated. Here, both intracellular growth of Legionella pneumophila (luminescence) and host cell viability (SYTOX Green exclusion) were assayed in the same screening well. Incorporation of membrane-impermeant, DNA-binding, fluorescent dyes in HTS assays should prove useful by allowing evaluation of cytotoxicity in real time, eliminating reagent addition steps and effort associated with endpoint cell viability analysis, and reducing the need for follow-up cytotoxicity screening. PMID:24831788

Real-Time Alpine Measurement System Using Wireless Sensor Networks

PubMed Central

2017-01-01

Monitoring the snow pack is crucial for many stakeholders, whether for hydro-power optimization, water management or flood control. Traditional forecasting relies on regression methods, which often results in snow melt runoff predictions of low accuracy in non-average years. Existing ground-based real-time measurement systems do not cover enough physiographic variability and are mostly installed at low elevations. We present the hardware and software design of a state-of-the-art distributed Wireless Sensor Network (WSN)-based autonomous measurement system with real-time remote data transmission that gathers data of snow depth, air temperature, air relative humidity, soil moisture, soil temperature, and solar radiation in physiographically representative locations. Elevation, aspect, slope and vegetation are used to select network locations, and distribute sensors throughout a given network location, since they govern snow pack variability at various scales. Three WSNs were installed in the Sierra Nevada of Northern California throughout the North Fork of the Feather River, upstream of the Oroville dam and multiple powerhouses along the river. The WSNs gathered hydrologic variables and network health statistics throughout the 2017 water year, one of northern Sierra’s wettest years on record. These networks leverage an ultra-low-power wireless technology to interconnect their components and offer recovery features, resilience to data loss due to weather and wildlife disturbances and real-time topological visualizations of the network health. Data show considerable spatial variability of snow depth, even within a 1 km2 network location. Combined with existing systems, these WSNs can better detect precipitation timing and phase in, monitor sub-daily dynamics of infiltration and surface runoff during precipitation or snow melt, and inform hydro power managers about actual ablation and end-of-season date across the landscape. PMID:29120376

Real-Time Alpine Measurement System Using Wireless Sensor Networks.

PubMed

Malek, Sami A; Avanzi, Francesco; Brun-Laguna, Keoma; Maurer, Tessa; Oroza, Carlos A; Hartsough, Peter C; Watteyne, Thomas; Glaser, Steven D

2017-11-09

Monitoring the snow pack is crucial for many stakeholders, whether for hydro-power optimization, water management or flood control. Traditional forecasting relies on regression methods, which often results in snow melt runoff predictions of low accuracy in non-average years. Existing ground-based real-time measurement systems do not cover enough physiographic variability and are mostly installed at low elevations. We present the hardware and software design of a state-of-the-art distributed Wireless Sensor Network (WSN)-based autonomous measurement system with real-time remote data transmission that gathers data of snow depth, air temperature, air relative humidity, soil moisture, soil temperature, and solar radiation in physiographically representative locations. Elevation, aspect, slope and vegetation are used to select network locations, and distribute sensors throughout a given network location, since they govern snow pack variability at various scales. Three WSNs were installed in the Sierra Nevada of Northern California throughout the North Fork of the Feather River, upstream of the Oroville dam and multiple powerhouses along the river. The WSNs gathered hydrologic variables and network health statistics throughout the 2017 water year, one of northern Sierra's wettest years on record. These networks leverage an ultra-low-power wireless technology to interconnect their components and offer recovery features, resilience to data loss due to weather and wildlife disturbances and real-time topological visualizations of the network health. Data show considerable spatial variability of snow depth, even within a 1 km 2 network location. Combined with existing systems, these WSNs can better detect precipitation timing and phase in, monitor sub-daily dynamics of infiltration and surface runoff during precipitation or snow melt, and inform hydro power managers about actual ablation and end-of-season date across the landscape.

Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP).

PubMed

Yang, Sunny Y; Amor, Souheila; Laguerre, Aurélien; Wong, Judy M Y; Monchaud, David

2017-05-01

The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of time-resolved fluorescence for direct and continuous probing of release from polymeric delivery vehicles.

PubMed

Viger, Mathieu L; Sheng, Wangzhong; McFearin, Cathryn L; Berezin, Mikhail Y; Almutairi, Adah

2013-11-10

Though accurately evaluating the kinetics of release is critical for validating newly designed therapeutic carriers for in vivo applications, few methods yet exist for release measurement in real time and without the need for any sample preparation. Many of the current approaches (e.g. chromatographic methods, absorption spectroscopy, or NMR spectroscopy) rely on isolation of the released material from the loaded vehicles, which require additional sample purification and can lead to loss of accuracy when probing fast kinetics of release. In this study we describe the use of time-resolved fluorescence for in situ monitoring of small molecule release kinetics from biodegradable polymeric drug delivery systems. This method relies on the observation that fluorescent reporters being released from polymeric drug delivery systems possess distinct excited-state lifetime components, reflecting their different environments in the particle suspensions, i.e., confined in the polymer matrices or free in the aqueous environment. These distinct lifetimes enable real-time quantitative mapping of the relative concentrations of dye in each population to obtain precise and accurate temporal information on the release profile of particular carrier/payload combinations. We found that fluorescence lifetime better distinguishes subtle differences in release profiles (e.g. differences associated with dye loading) than conventional steady-state fluorescence measurements, which represent the averaged dye behavior over the entire scan. Given the method's applicability to both hydrophobic and hydrophilic cargo, it could be employed to model the release of any drug-carrier combination. Copyright © 2013 Elsevier B.V. All rights reserved.

Real-Time Nanoscopy by Using Blinking Enhanced Quantum Dots

PubMed Central

Watanabe, Tomonobu M.; Fukui, Shingo; Jin, Takashi; Fujii, Fumihiko; Yanagida, Toshio

2010-01-01

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices. PMID:20923631

Comparison of three-way and four-way calibration for the real-time quantitative analysis of drug hydrolysis in complex dynamic samples by excitation-emission matrix fluorescence.

PubMed

Yin, Xiao-Li; Gu, Hui-Wen; Liu, Xiao-Lu; Zhang, Shan-Hui; Wu, Hai-Long

2018-03-05

Multiway calibration in combination with spectroscopic technique is an attractive tool for online or real-time monitoring of target analyte(s) in complex samples. However, how to choose a suitable multiway calibration method for the resolution of spectroscopic-kinetic data is a troubling problem in practical application. In this work, for the first time, three-way and four-way fluorescence-kinetic data arrays were generated during the real-time monitoring of the hydrolysis of irinotecan (CPT-11) in human plasma by excitation-emission matrix fluorescence. Alternating normalization-weighted error (ANWE) and alternating penalty trilinear decomposition (APTLD) were used as three-way calibration for the decomposition of the three-way kinetic data array, whereas alternating weighted residual constraint quadrilinear decomposition (AWRCQLD) and alternating penalty quadrilinear decomposition (APQLD) were applied as four-way calibration to the four-way kinetic data array. The quantitative results of the two kinds of calibration models were fully compared from the perspective of predicted real-time concentrations, spiked recoveries of initial concentration, and analytical figures of merit. The comparison study demonstrated that both three-way and four-way calibration models could achieve real-time quantitative analysis of the hydrolysis of CPT-11 in human plasma under certain conditions. However, it was also found that both of them possess some critical advantages and shortcomings during the process of dynamic analysis. The conclusions obtained in this paper can provide some helpful guidance for the reasonable selection of multiway calibration models to achieve the real-time quantitative analysis of target analyte(s) in complex dynamic systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of tissue optical properties on time-resolved fluorescence measurements from brain tumors: an experimental and computational study

NASA Astrophysics Data System (ADS)

Butte, Pramod V.; Vishwanath, Karthik; Pikul, Brian K.; Mycek, Mary-Ann; Marcu, Laura

2003-07-01

Time-Resolved Laser-Induced Fluorescence Spectroscopy (tr-LIFS) offers the potential for intra-operative diagnosis of primary brain tumors. However, both the intrinsic properties of endogenous fluorophores and the optical properties of brain tissue could affect the fluorescence measurements from brain. Scattering has been demonstrated to increase, for instance, detected lifetimes by 10-20% in media less scattering than the brain. The overall goal of this study is to investigate experimentally and computationally how optical properties of distinct types of brain tissue (normal porcine white and gray matter) affect the propagation of the excitation pulse and fluorescent transients and the detected fluorescence lifetime. A time-domain tr-LIFS apparatus (fast digitizer and gated detection) was employed to measure the propagation of ultra-short pulsed light through brain specimens (1-2.5-mm source-detector separation; 0.100-mm increment). A Monte Carlo model for semi-infinite turbid media was used to simulate time-resolved light propagation for arbitrary source-detector fiber geometries and optical fiber specifications; and to record spatially- and temporally resolved information. We determined a good correlation between experimental and computational results. Our findings provide means for quantification of time-resolved fluorescence spectra from healthy and diseased brain tissue.

Real-Time Protein and Cell Binding Measurements on Hydroxyapatite Coatings

PubMed Central

Vilardell, A. M.; Cinca, N.; Jokinen, A.; Garcia-Giralt, N.; Dosta, S.; Cano, I. G.; Guilemany, J. M.

2016-01-01

Although a lot of in vitro and in vivo assays have been performed during the last few decades years for hydroxyapatite bioactive coatings, there is a lack of exploitation of real-time in vitro interaction measurements. In the present work, real-time interactions for a plasma sprayed hydroxyapatite coating were measured by a Multi-Parametric Surface Plasmon Resonance (MP-SPR), and the results were compared with standard traditional cell viability in vitro assays. MP-SPR is proven to be suitable not only for measurement of molecule–molecule interactions but also molecule–material interaction measurements and cell interaction. Although SPR is extensively utilized in interaction studies, recent research of protein or cell adsorption on hydroxyapatite coatings for prostheses applications was not found. The as-sprayed hydroxyapatite coating resulted in 62.4% of crystalline phase and an average thickness of 24 ± 6 μm. The MP-SPR was used to measure lysozyme protein and human mesenchymal stem cells interaction to the hydroxyapatite coating. A comparison between the standard gold sensor and Hydroxyapatite (HA)-plasma coated sensor denoted a clearly favourable cell attachment on HA coated sensor as a significantly higher signal of cell binding was detected. Moreover, traditional cell viability and proliferation tests showed increased activity with culture time indicating that cells were proliferating on HA coating. Cells show homogeneous distribution and proliferation along the HA surface between one and seven days with no significant mortality. Cells were flattened and spread on rough surfaces from the first day, with increasing cytoplasmatic extensions during the culture time. PMID:27618911

Femtosecond/picosecond time-resolved fluorescence study of hydrophilic polymer fine particles.

PubMed

Nanjo, Daisuke; Hosoi, Haruko; Fujino, Tatsuya; Tahara, Tahei; Korenaga, Takashi

2007-03-22

Femtosecond/picosecond time-resolved fluorescence study of hydrophilic polymer fine particles (polyacrylamide, PAAm) was reported. Ultrafast fluorescence dynamics of polymer/water solution was monitored using a fluorescent probe molecule (C153). In the femtosecond time-resolved fluorescence measurement at 480 nm, slowly decay components having lifetimes of tau(1) approximately 53 ps and tau(2) approximately 5 ns were observed in addition to rapid fluorescence decay. Picosecond time-resolved fluorescence spectra of C153/PAAm/H2O solution were also measured. In the time-resolved fluorescence spectra of C153/PAAm/H2O, a peak shift from 490 to 515 nm was measured, which can be assigned to the solvation dynamics of polymer fine particles. The fluorescence peak shift was related to the solvation response function and two time constants were determined (tau(3) approximately 50 ps and tau(4) approximately 467 ps). Therefore, the tau(1) component observed in the femtosecond time-resolved fluorescence measurement was assigned to the solvation dynamics that was observed only in the presence of polymer fine particles. Rotational diffusion measurements were also carried out on the basis of the picosecond time-resolved fluorescence spectra. In the C153/PAAm/H2O solution, anisotropy decay having two different time constants was also derived (tau(6) approximately 76 ps and tau(7) approximately 676 ps), indicating the presence of two different microscopic molecular environments around the polymer surface. Using the Stokes-Einstein-Debye (SED) equation, microscopic viscosity around the polymer surface was evaluated. For the area that gave a rotational diffusion time of tau(6) approximately 76 ps, the calculated viscosity is approximately 1.1 cP and for tau(7) approximately 676 ps, it is approximately 10 cP. The calculated viscosity values clearly revealed that there are two different molecular environments around the polyacrylamide fine particles.

Dynamic fiber Bragg grating strain sensor interrogation with real-time measurement

NASA Astrophysics Data System (ADS)

Park, Jinwoo; Kwon, Yong Seok; Ko, Myeong Ock; Jeon, Min Yong

2017-11-01

We demonstrate a 1550 nm band resonance Fourier-domain mode-locked (FDML) fiber laser with fiber Bragg grating (FBG) array. Using the FDML fiber laser, we successfully demonstrate real-time monitoring of dynamic FBG strain sensor interrogation for structural health monitoring. The resonance FDML fiber laser consists of six multiplexed FBGs, which are arranged in series with delay fiber lengths. It is operated by driving the fiber Fabry-Perot tunable filter (FFP-TF) with a sinusoidal waveform at a frequency corresponding to the round-trip time of the laser cavity. Each FBG forms a laser cavity independently in the FDML fiber laser because the light travels different length for each FBG. The very closely positioned two FBGs in a pair are operated simultaneously with a frequency in the FDML fiber laser. The spatial positions of the sensing pair can be distinguished from the variation of the applied frequency to the FFP-TF. One of the FBGs in the pair is used as a reference signal and the other one is fixed on the piezoelectric transducer stack to apply the dynamic strain. We successfully achieve real-time measurement of the abrupt change of the frequencies applied to the FBG without any signal processing delay. The real-time monitoring system is displayed simultaneously on the monitor for the variation of the two peaks, the modulation interval of the two peaks, and their fast Fourier transform spectrum. The frequency resolution of the dynamic variation could reach up to 0.5 Hz for 2 s integration time. It depends on the integration time to measure the dynamic variation. We believe that the real-time monitoring system will have a potential application for structural health monitoring.

Measuring inhibition of monoamine reuptake transporters by new psychoactive substances (NPS) in real-time using a high-throughput, fluorescence-based assay.

PubMed

Zwartsen, Anne; Verboven, Anouk H A; van Kleef, Regina G D M; Wijnolts, Fiona M J; Westerink, Remco H S; Hondebrink, Laura

2017-12-01

The prevalence and use of new psychoactive substances (NPS) is increasing and currently over 600 NPS exist. Many illicit drugs and NPS increase brain monoamine levels by inhibition and/or reversal of monoamine reuptake transporters (DAT, NET and SERT). This is often investigated using labor-intensive, radiometric endpoint measurements. We investigated the applicability of a novel and innovative assay that is based on a fluorescent monoamine mimicking substrate. DAT, NET or SERT-expressing human embryonic kidney (HEK293) cells were exposed to common drugs (cocaine, dl-amphetamine or MDMA), NPS (4-fluoroamphetamine, PMMA, α-PVP, 5-APB, 2C-B, 25B-NBOMe, 25I-NBOMe or methoxetamine) or the antidepressant fluoxetine. We demonstrate that this fluorescent microplate reader-based assay detects inhibition of different transporters by various drugs and discriminates between drugs. Most IC 50 values were in line with previous results from radiometric assays and within estimated human brain concentrations. However, phenethylamines showed higher IC 50 values on hSERT, possibly due to experimental differences. Compared to radiometric assays, this high-throughput fluorescent assay is uncomplicated, can measure at physiological conditions, requires no specific facilities and allows for kinetic measurements, enabling detection of transient effects. This assay is therefore a good alternative for radiometric assays to investigate effects of illicit drugs and NPS on monoamine reuptake transporters. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Real-Time Unsteady Loads Measurements Using Hot-Film Sensors

NASA Technical Reports Server (NTRS)

Mangalam, Arun S.; Moes, Timothy R.

2004-01-01

Several flight-critical aerodynamic problems such as buffet, flutter, stall, and wing rock are strongly affected or caused by abrupt changes in unsteady aerodynamic loads and moments. Advanced sensing and flow diagnostic techniques have made possible simultaneous identification and tracking, in real-time, of the critical surface, viscosity-related aerodynamic phenomena under both steady and unsteady flight conditions. The wind tunnel study reported here correlates surface hot-film measurements of leading edge stagnation point and separation point, with unsteady aerodynamic loads on a NACA 0015 airfoil. Lift predicted from the correlation model matches lift obtained from pressure sensors for an airfoil undergoing harmonic pitchup and pitchdown motions. An analytical model was developed that demonstrates expected stall trends for pitchup and pitchdown motions. This report demonstrates an ability to obtain unsteady aerodynamic loads in real-time, which could lead to advances in air vehicle safety, performance, ride-quality, control, and health management.

In-situ real time measurements of net erosion rates of copper during hydrogen plasma exposure

NASA Astrophysics Data System (ADS)

Kesler, Leigh; Wright, Graham; Peterson, Ethan; Whyte, Dennis

2013-10-01

In order to properly understand the dynamics of net erosion/deposition in fusion reactors, such as tokamaks, a diagnostic measuring the real time rates of net erosion/deposition during plasma exposure is necessary. The DIONISOS experiment produces real time measurements of net erosion/deposition by using Rutherford backscattering spectroscopy (RBS) ion beam analysis simultaneously with plasma exposure from a helicon plasma source. This in-situ method improves on ex-situ weight loss measurements by allowing measurement of possible synergistic effects of high ion implantation rates and net erosion rate and by giving a real time response to changes in plasma parameters. Previous work has validated this new technique for measuring copper (Cu) erosion from helium (He) plasma ion bombardment. This technique is now extended to measure copper erosion due to deuterium and hydrogen plasma ion exposure. Targets used were a 1.5 μm Cu layer on an aluminum substrate. Cu layer thickness is tracked in real time using 1.2 MeV proton RBS. Measured erosion rates will be compared to results from literature and He erosion rates. Supported by US DoE award DE-SC00-02060.

An automated real-time microscopy system for analysis of fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Bernardini, André; Wotzlaw, Christoph; Lipinski, Hans-Gerd; Fandrey, Joachim

2010-05-01

Molecular imaging based on Fluorescence Resonance Energy Transfer (FRET) is widely used in cellular physiology both for protein-protein interaction analysis and detecting conformational changes of single proteins, e.g. during activation of signaling cascades. However, getting reliable results from FRET measurements is still hampered by methodological problems such as spectral bleed through, chromatic aberration, focal plane shifts and false positive FRET. Particularly false positive FRET signals caused by random interaction of the fluorescent dyes can easily lead to misinterpretation of the data. This work introduces a Nipkow Disc based FRET microscopy system, that is easy to operate without expert knowledge of FRET. The system automatically accounts for all relevant sources of errors and provides various result presentations of two, three and four dimensional FRET data. Two examples are given to demonstrate the scope of application. An interaction analysis of the two subunits of the hypoxia-inducible transcription factor 1 demonstrates the use of the system as a tool for protein-protein interaction analysis. As an example for time lapse observations, the conformational change of the fluorophore labeled heat shock protein 33 in the presence of oxidant stress is shown.

Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments.

PubMed

Montgomery, Jesse L; Wittwer, Carl T

2014-02-01

Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30-90 min. The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1-100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%-90% activated after 20 min. Simple DNA extension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations, DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR.

Fluorescent microscope system to monitor real-time interactions between focused ultrasound, echogenic drug delivery vehicles, and live cell membranes.

PubMed

Ibsen, Stuart; Benchimol, Michael; Esener, Sadik

2013-01-01

Rapid development in the field of ultrasound triggered drug delivery has made it essential to study the real-time interaction between the membranes of live cells and the membranes of echogenic delivery vehicles under exposure to focused ultrasound. The objective of this work was to design an analysis system that combined fluorescent imagining, high speed videography, and definable pulse sequences of focused ultrasound to allow for real time observations of both cell and vehicle membranes. Documenting the behavior of the membranes themselves has not previously been possible due to limitations with existing optical systems used to understand the basic physics of microbubble/ultrasound interaction and the basic interaction between microbubbles and cells. The performance of this new system to monitor membrane behavior was demonstrated by documenting the modes of vehicle fragmentation at different ultrasound intensity levels. At 1.5MPa the membranes were shown to completely fragment while at intensities below 1MPa the membranes pop open and slowly unfold. The interaction between these vehicles and cell membranes was also documented by the removal of fluorescent particles from the surfaces of live cells out to 20μm from the microbubble location. The fluid flow created by microstreaming around ensonated microbubbles was documented at video recording speeds from 60 to 18,000 frames per second. This information about membrane behavior allows the chemical and physical properties of the drug delivery vehicle to be designed along with the ultrasound pulse sequence to cause the most efficient drug delivery. Copyright © 2012 Elsevier B.V. All rights reserved.

Fluorescent Microscope System to Monitor Real-Time Interactions between Focused Ultrasound, Echogenic Drug Delivery Vehicles, and Live Cell Membranes

PubMed Central

Ibsen, Stuart; Benchimol, Michael; Esener, Sadik

2012-01-01

Rapid development in the field of ultrasound triggered drug delivery has made it essential to study the real-time interaction between the membranes of live cells and the membranes of echogenic delivery vehicles under exposure to focused ultrasound. The objective of this work was to design an analysis system that combined fluorescent imagining, high speed videography, and definable pulse sequences of focused ultrasound to allow for real time observations of both cell and vehicle membranes. Documenting the behavior of the membranes themselves has not previously been possible due to limitations with existing optical systems used to understand the basic physics of microbubble/ultrasound interaction and the basic interaction between microbubbles and cells. The performance of this new system to monitor membrane behavior was demonstrated by documenting the modes of vehicle fragmentation at different ultrasound intensity levels. At 1.5 MPa the membranes were shown to completely fragment while at intensities below 1 MPa there is a popping and slow unfolding. The interaction between these vehicles and cell membranes was also documented by the removal of fluorescent particles from the surfaces of live cells out to 20 μm from the microbubble location. The fluid flow created by microstreaming around ensonated microbubbles was documented at video recording speeds from 60 to 18,000 frames per second. This information about membrane behavior allows the chemical and physical properties of the drug delivery vehicle to be designed along with the ultrasound pulse sequence to cause the most efficient drug delivery. PMID:22749476

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

PubMed Central

2011-01-01

Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested. Results Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA. Conclusions The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings. PMID:21851640

Miniaturized and Wireless Optical Neurotransmitter Sensor for Real-Time Monitoring of Dopamine in the Brain

PubMed Central

Kim, Min H.; Yoon, Hargsoon; Choi, Sang H.; Zhao, Fei; Kim, Jongsung; Song, Kyo D.; Lee, Uhn

2016-01-01

Real-time monitoring of extracellular neurotransmitter concentration offers great benefits for diagnosis and treatment of neurological disorders and diseases. This paper presents the study design and results of a miniaturized and wireless optical neurotransmitter sensor (MWONS) for real-time monitoring of brain dopamine concentration. MWONS is based on fluorescent sensing principles and comprises a microspectrometer unit, a microcontroller for data acquisition, and a Bluetooth wireless network for real-time monitoring. MWONS has a custom-designed application software that controls the operation parameters for excitation light sources, data acquisition, and signal processing. MWONS successfully demonstrated a measurement capability with a limit of detection down to a 100 nanomole dopamine concentration, and high selectivity to ascorbic acid (90:1) and uric acid (36:1). PMID:27834927

Miniaturized and Wireless Optical Neurotransmitter Sensor for Real-Time Monitoring of Dopamine in the Brain.

PubMed

Kim, Min H; Yoon, Hargsoon; Choi, Sang H; Zhao, Fei; Kim, Jongsung; Song, Kyo D; Lee, Uhn

2016-11-10

Real-time monitoring of extracellular neurotransmitter concentration offers great benefits for diagnosis and treatment of neurological disorders and diseases. This paper presents the study design and results of a miniaturized and wireless optical neurotransmitter sensor (MWONS) for real-time monitoring of brain dopamine concentration. MWONS is based on fluorescent sensing principles and comprises a microspectrometer unit, a microcontroller for data acquisition, and a Bluetooth wireless network for real-time monitoring. MWONS has a custom-designed application software that controls the operation parameters for excitation light sources, data acquisition, and signal processing. MWONS successfully demonstrated a measurement capability with a limit of detection down to a 100 nanomole dopamine concentration, and high selectivity to ascorbic acid (90:1) and uric acid (36:1).

Measuring the complexity of design in real-time imaging software

NASA Astrophysics Data System (ADS)

Sangwan, Raghvinder S.; Vercellone-Smith, Pamela; Laplante, Phillip A.

2007-02-01

Due to the intricacies in the algorithms involved, the design of imaging software is considered to be more complex than non-image processing software (Sangwan et al, 2005). A recent investigation (Larsson and Laplante, 2006) examined the complexity of several image processing and non-image processing software packages along a wide variety of metrics, including those postulated by McCabe (1976), Chidamber and Kemerer (1994), and Martin (2003). This work found that it was not always possible to quantitatively compare the complexity between imaging applications and nonimage processing systems. Newer research and an accompanying tool (Structure 101, 2006), however, provides a greatly simplified approach to measuring software complexity. Therefore it may be possible to definitively quantify the complexity differences between imaging and non-imaging software, between imaging and real-time imaging software, and between software programs of the same application type. In this paper, we review prior results and describe the methodology for measuring complexity in imaging systems. We then apply a new complexity measurement methodology to several sets of imaging and non-imaging code in order to compare the complexity differences between the two types of applications. The benefit of such quantification is far reaching, for example, leading to more easily measured performance improvement and quality in real-time imaging code.

Genetically encoded sensors enable real-time observation of metabolite production

PubMed Central

Rogers, Jameson K.; Church, George M.

2016-01-01

Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules. PMID:26858408

Genetically encoded sensors enable real-time observation of metabolite production

DOE PAGES

Rogers, Jameson K.; Church, George M.

2016-02-08

Here, engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allowsmore » us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of 14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of 51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules.« less

Real-time point-of-care measurement of impaired renal function in a rat acute injury model employing exogenous fluorescent tracer agents

NASA Astrophysics Data System (ADS)

Dorshow, Richard B.; Fitch, Richard M.; Galen, Karen P.; Wojdyla, Jolette K.; Poreddy, Amruta R.; Freskos, John N.; Rajagopalan, Raghavan; Shieh, Jeng-Jong; Demirjian, Sevag G.

2013-02-01

Renal function assessment is needed for the detection of acute kidney injury and chronic kidney disease. Glomerular filtration rate (GFR) is now widely accepted as the best indicator of renal function, and current clinical guidelines advocate its use in the staging of kidney disease. The optimum measure of GFR is by the use of exogenous tracer agents. However current clinically employed agents lack sensitivity or are cumbersome to use. An exogenous GFR fluorescent tracer agent, whose elimination rate could be monitored noninvasively through skin would provide a substantial improvement over currently available methods. We developed a series of novel aminopyrazine analogs for use as exogenous fluorescent GFR tracer agents that emit light in the visible region for monitoring GFR noninvasively over skin. In rats, these compounds are eliminated by the kidney with urine recovery greater than 90% of injected dose, are not broken down or metabolized in vivo, are not secreted by the renal tubules, and have clearance values similar to a GFR reference compound, iothalamate. In addition, biological half-life of these compounds measured in rats by noninvasive optical methods correlated with plasma derived methods. In this study, we show that this noninvasive methodology with our novel fluorescent tracer agents can detect impaired renal function. A 5/6th nephrectomy rat model is employed.

A real-time fluorescent sensor specific to Mg2+: crystallographic evidence, DFT calculation and its use for quantitative determination of magnesium in drinking water.

PubMed

Men, Guangwen; Chen, Chunrong; Zhang, Shitong; Liang, Chunshuang; Wang, Ying; Deng, Mengyu; Shang, Hongxing; Yang, Bing; Jiang, Shimei

2015-02-14

An "off-the-shelf" fluorescence "turn-on" Mg(2+) chemosensor 3,5-dichlorosalicylaldehyde (BCSA) was rationally designed and developed. This proposed sensor works based on Mg(2+)-induced formation of the 2 : 1 BCSA-Mg(2+) complex. The coordination of BSCA to Mg(2+) increases its structural rigidity generating a chelation-enhanced fluorescence (CHEF) effect which was confirmed by single crystal XRD studies of the BSCA-Mg(2+) complex and TD/DFT calculations. This sensor exhibits high sensitivity and selectivity for the quantitative monitoring of Mg(2+) with a wide detection range (0-40 μM), a low detection limit (2.89 × 10(-7) mol L(-1)) and a short response time (<0.5 s). It can also resist the interference from the other co-existing metal ions, especially Ca(2+). Consequently, this fluorescent sensor can be utilized to monitor Mg(2+) in real time within actual samples from drinking water.

Digital Holography for in Situ Real-Time Measurement of Plasma-Facing-Component Erosion

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

ThomasJr., C. E.; Granstedt, E. M.; Biewer, Theodore M

2014-01-01

In situ, real time measurement of net plasma-facing-component (PFC) erosion/deposition in a real plasma device is challenging due to the need for good spatial and temporal resolution, sufficient sensitivity, and immunity to fringe-jump errors. Design of a high-sensitivity, potentially high-speed, dual-wavelength CO2 laser digital holography system (nominally immune to fringe jumps) for PFC erosion measurement is discussed.

Direct real-time measurement of intra-oocyte nitric oxide concentration in vivo.

PubMed

Goud, Pravin T; Goud, Anuradha P; Najafi, Tohid; Gonik, Bernard; Diamond, Michael P; Saed, Ghassan M; Zhang, Xueji; Abu-Soud, Husam M

2014-01-01

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70-75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process.

Direct Real-Time Measurement of Intra-Oocyte Nitric Oxide Concentration In Vivo

PubMed Central

Goud, Pravin T.; Goud, Anuradha P.; Najafi, Tohid; Gonik, Bernard; Diamond, Michael P.; Saed, Ghassan M.; Zhang, Xueji; Abu-Soud, Husam M.

2014-01-01

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70–75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process. PMID:24887331

Real-time 3D measurement based on structured light illumination considering camera lens distortion

NASA Astrophysics Data System (ADS)

Feng, Shijie; Chen, Qian; Zuo, Chao; Sun, Jiasong; Yu, ShiLing

2014-12-01

Optical three-dimensional (3-D) profilometry is gaining increasing attention for its simplicity, flexibility, high accuracy, and non-contact nature. Recent advances in imaging sensors and digital projection technology further its progress in high-speed, real-time applications, enabling 3-D shapes reconstruction of moving objects and dynamic scenes. In traditional 3-D measurement system where the processing time is not a key factor, camera lens distortion correction is performed directly. However, for the time-critical high-speed applications, the time-consuming correction algorithm is inappropriate to be performed directly during the real-time process. To cope with this issue, here we present a novel high-speed real-time 3-D coordinates measuring technique based on fringe projection with the consideration of the camera lens distortion. A pixel mapping relation between a distorted image and a corrected one is pre-determined and stored in computer memory for real-time fringe correction. And a method of lookup table (LUT) is introduced as well for fast data processing. Our experimental results reveal that the measurement error of the in-plane coordinates has been reduced by one order of magnitude and the accuracy of the out-plane coordinate been tripled after the distortions being eliminated. Moreover, owing to the merit of the LUT, the 3-D reconstruction can be achieved at 92.34 frames per second.

Real time cancer prediction based on objective tissue compliance measurement in endoscopic surgery.

PubMed

Fakhry, Morkos; Bello, Fernando; Hanna, George B

2014-02-01

To investigate the feasibility of real time cancer tissue diagnosis intraoperatively based on in vivo tissue compliance measurements obtained by a recently developed laparoscopic smart device. Cancer tissue is stiffer than its normal counterpart. Modern forms of remote surgery such as laparoscopic and robotic surgical techniques diminish direct assessment of this important tissue property. In vivo human tissue compliance of the normal and cancer gastrointestinal tissue is unknown. A Clinical Real Time Tissue Compliance Mapping System (CRTCMS) with a predictive power comparable to the human hand and useable in routine surgical practice has been recently developed. The CRTCMS is employed in the operating theater to collect data from 50 patients undergoing intra-abdominal surgical interventions [40 men, 10 women, aged between 32 and 89 (mean = 66.4, range = 57)]. This includes 10 esophageal and 27 gastric cancer patients. A total of 1212 compliance measurements of normal and cancerous in vivo gastrointestinal tissues were taken. The data were used to calibrate the CRTCMS to predict cancerous tissue in a further 12 patients (3 cancer esophagus and 9 cancer stomach) involving 175 measurements. The system demonstrated a high prediction power to diagnose cancer tissue in real time during routine surgical procedures (sensitivity = 98.7%, specificity = 99%). An in vivo human tissue compliance data bank of the gastrointestinal tract was produced. Real time cancer diagnosis based on in vivo tissue compliance measurements is feasible. The reported data open new avenues in cancer diagnostics, surgical robotics, and development of more realistic surgical simulators.

Real-time control of sewer systems using turbidity measurements.

PubMed

Lacour, C; Schütze, M

2011-01-01

Real-time control (RTC) of urban drainage systems has been proven useful as a means to reduce pollution by combined sewer overflow discharges. So far, RTC has been investigated mainly with a sole focus on water quantity aspects. However, as measurement techniques for pollution of wastewater are advancing, pollution-based RTC might be of increasing interest. For example, turbidity data sets from an extensive measurement programme in two Paris catchments allow a detailed investigation of the benefits of using pollution-based data for RTC. This paper exemplifies this, comparing pollution-based RTC with flow-based RTC. Results suggest that pollution-based RTC indeed has some potential, particularly when measurements of water-quality characteristics are readily available.

IMU-based Real-time Pose Measurement system for Anterior Pelvic Plane in Total Hip Replacement Surgeries.

PubMed

Zhe Cao; Shaojie Su; Hao Tang; Yixin Zhou; Zhihua Wang; Hong Chen

2017-07-01

With the aging of population, the number of Total Hip Replacement Surgeries (THR) increased year by year. In THR, inaccurate position of the implanted prosthesis may lead to the failure of the operation. In order to reduce the failure rate and acquire the real-time pose of Anterior Pelvic Plane (APP), we propose a measurement system in this paper. The measurement system includes two parts: Initial Pose Measurement Instrument (IPMI) and Real-time Pose Measurement Instrument (RPMI). IPMI is used to acquire the initial pose of the APP, and RPMI is used to estimate the real-time pose of the APP. Both are composed of an Inertial Measurement Unit (IMU) and magnetometer sensors. To estimate the attitude of the measurement system, the Extended Kalman Filter (EKF) is adopted in this paper. The real-time pose of the APP could be acquired together with the algorithm designed in the paper. The experiment results show that the Root Mean Square Error (RMSE) is within 1.6 degrees, which meets the requirement of THR operations.

Real-time Kp predictions from ACE real time solar wind

NASA Astrophysics Data System (ADS)

Detman, Thomas; Joselyn, Joann

1999-06-01

The Advanced Composition Explorer (ACE) spacecraft provides nearly continuous monitoring of solar wind plasma, magnetic fields, and energetic particles from the Sun-Earth L1 Lagrange point upstream of Earth in the solar wind. The Space Environment Center (SEC) in Boulder receives ACE telemetry from a group of international network of tracking stations. One-minute, and 1-hour averages of solar wind speed, density, temperature, and magnetic field components are posted on SEC's World Wide Web page within 3 to 5 minutes after they are measured. The ACE Real Time Solar Wind (RTSW) can be used to provide real-time warnings and short term forecasts of geomagnetic storms based on the (traditional) Kp index. Here, we use historical data to evaluate the performance of the first real-time Kp prediction algorithm to become operational.

Micro-scale temperature measurement method using fluorescence polarization

NASA Astrophysics Data System (ADS)

Tatsumi, K.; Hsu, C.-H.; Suzuki, A.; Nakabe, K.

2016-09-01

A novel method that can measure the fluid temperature in microscopic scale by measuring the fluorescence polarization is described in this paper. The measurement technique is not influenced by the quenching effects which appears in conventional LIF methods and is believed to show a higher reliability in temperature measurements. Experiment was performed using a microchannel flow and fluorescent molecule probes, and the effects of the fluid temperature, fluid viscosity, measurement time, and pH of the solution on the measured fluorescence polarization degree are discussed to understand the basic characteristics of the present method. The results showed that fluorescence polarization is considerably less sensible to these quenching factors. A good correlation with the fluid temperature, on the other hand, was obtained and agreed well with the theoretical values confirming the feasibility of the method.

Measuring Photosynthetic Response to Drought Stress using Active and Passive Fluorescence

NASA Astrophysics Data System (ADS)

Helm, L.; Lerdau, M.; Wang, W.; Yang, X.

2017-12-01

Photosynthesis, the endothermic reactions involving the absorption of light and fixation and reduction of carbon dioxide by plants, plays important roles in carbon and water cycles, food security, and even weather and climate patterns. Solar radiation provides the energy for photosynthesis, but often plants absorb more solar energy than they can use to reduce carbon dioxide. This excess energy, which is briefly stored as high-energy electrons in the chloroplast, must be removed or damage to the leaf's photosynthetic machinery will occur. One important energy dissipation pathway is for the high energy electrons to return to their lower valance state and, in doing so, release radiation (fluorescence). This fluorescence (known as solar induced fluorescence (SIF) has been found to strongly correlate with gross photosynthesis. Recent advances in the remote sensing of SIF allow for large-scale real-time estimation of photosynthesis. In a warming climate with more frequent stress, remote sensing is necessary for measuring the spatial and temporal variability of photosynthesis. However, the mechanisms that link SIF and photosynthesis are unclear, particularly how the relationship may or may not change under stress. We present data from leaf-level measurements of gas exchange, pulse amplitude modulation (PAM) fluorescence, and SIF in two major tree species in North America. Water-stressed and well-watered plants were compared to determine how SIF and carbon dioxide exchange are modulated by drought diurnally and seasonally. Secondly, photosynthesis and fluorescence under high and low oxygen concentrations were compared to determine how photorespiration alters the relationship between SIF and gross photosynthesis. We find a strong correlation between SIF and steady-state fluorescence measured with conventional PAM fluorometry. Our results also indicate that drought-stress modulates the SIF-photosynthesis relationship, and this may be driven by drought-induced changes in

Method and apparatus for real-time measurement of fuel gas compositions and heating values

DOEpatents

Zelepouga, Serguei; Pratapas, John M.; Saveliev, Alexei V.; Jangale, Vilas V.

2016-03-22

An exemplary embodiment can be an apparatus for real-time, in situ measurement of gas compositions and heating values. The apparatus includes a near infrared sensor for measuring concentrations of hydrocarbons and carbon dioxide, a mid infrared sensor for measuring concentrations of carbon monoxide and a semiconductor based sensor for measuring concentrations of hydrogen gas. A data processor having a computer program for reducing the effects of cross-sensitivities of the sensors to components other than target components of the sensors is also included. Also provided are corresponding or associated methods for real-time, in situ determination of a composition and heating value of a fuel gas.

Real-Time Aerodynamic Parameter Estimation without Air Flow Angle Measurements

NASA Technical Reports Server (NTRS)

Morelli, Eugene A.

2010-01-01

A technique for estimating aerodynamic parameters in real time from flight data without air flow angle measurements is described and demonstrated. The method is applied to simulated F-16 data, and to flight data from a subscale jet transport aircraft. Modeling results obtained with the new approach using flight data without air flow angle measurements were compared to modeling results computed conventionally using flight data that included air flow angle measurements. Comparisons demonstrated that the new technique can provide accurate aerodynamic modeling results without air flow angle measurements, which are often difficult and expensive to obtain. Implications for efficient flight testing and flight safety are discussed.

Detection of individual atoms in helium buffer gas and observation of their real-time motion

NASA Technical Reports Server (NTRS)

Pan, C. L.; Prodan, J. V.; Fairbank, W. M., Jr.; She, C. Y.

1980-01-01

Single atoms are detected and their motion measured for the first time to our knowledge by the fluorescence photon-burst method in the presence of large quantities of buffer gas. A single-clipped digital correlator records the photon burst in real time and displays the atom's transit time across the laser beam. A comparison is made of the special requirements for single-atom detection in vacuum and in a buffer gas. Finally, the probability distribution of the bursts from many atoms is measured. It further proves that the bursts observed on resonance are due to single atoms and not simply to noise fluctuations.

Accuracy of real time radiography burning rate measurement

NASA Astrophysics Data System (ADS)

Olaniyi, Bisola

The design of a solid propellant rocket motor requires the determination of a propellant's burning-rate and its dependency upon environmental parameters. The requirement that the burning-rate be physically measured, establishes the need for methods and equipment to obtain such data. A literature review reveals that no measurement has provided the desired burning rate accuracy. In the current study, flash x-ray modeling and digitized film-density data were employed to predict motor-port area to length ratio. The pre-fired port-areas and base burning rate were within 2.5% and 1.2% of their known values, respectively. To verify the accuracy of the method, a continuous x-ray and a solid propellant rocket motor model (Plexiglas cylinder) were used. The solid propellant motor model was translated laterally through a real-time radiography system at different speeds simulating different burning rates. X-ray images were captured and the burning-rate was then determined. The measured burning rate was within 1.65% of the known values.

Toward real-time virtual biopsy of oral lesions using confocal laser endomicroscopy interfaced with embedded computing.

PubMed

Thong, Patricia S P; Tandjung, Stephanus S; Movania, Muhammad Mobeen; Chiew, Wei-Ming; Olivo, Malini; Bhuvaneswari, Ramaswamy; Seah, Hock-Soon; Lin, Feng; Qian, Kemao; Soo, Khee-Chee

2012-05-01

Oral lesions are conventionally diagnosed using white light endoscopy and histopathology. This can pose a challenge because the lesions may be difficult to visualise under white light illumination. Confocal laser endomicroscopy can be used for confocal fluorescence imaging of surface and subsurface cellular and tissue structures. To move toward real-time "virtual" biopsy of oral lesions, we interfaced an embedded computing system to a confocal laser endomicroscope to achieve a prototype three-dimensional (3-D) fluorescence imaging system. A field-programmable gated array computing platform was programmed to enable synchronization of cross-sectional image grabbing and Z-depth scanning, automate the acquisition of confocal image stacks and perform volume rendering. Fluorescence imaging of the human and murine oral cavities was carried out using the fluorescent dyes fluorescein sodium and hypericin. Volume rendering of cellular and tissue structures from the oral cavity demonstrate the potential of the system for 3-D fluorescence visualization of the oral cavity in real-time. We aim toward achieving a real-time virtual biopsy technique that can complement current diagnostic techniques and aid in targeted biopsy for better clinical outcomes.

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids.

PubMed

Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

2008-06-04

Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

A Real-Time Wireless Sweat Rate Measurement System for Physical Activity Monitoring

PubMed Central

Brueck, Andrew; Iftekhar, Tashfin; Stannard, Alicja B.; Kaya, Tolga

2018-01-01

There has been significant research on the physiology of sweat in the past decade, with one of the main interests being the development of a real-time hydration monitor that utilizes sweat. The contents of sweat have been known for decades; sweat provides significant information on the physiological condition of the human body. However, it is important to know the sweat rate as well, as sweat rate alters the concentration of the sweat constituents, and ultimately affects the accuracy of hydration detection. Towards this goal, a calorimetric based flow-rate detection system was built and tested to determine sweat rate in real time. The proposed sweat rate monitoring system has been validated through both controlled lab experiments (syringe pump) and human trials. An Internet of Things (IoT) platform was embedded, with the sensor using a Simblee board and Raspberry Pi. The overall prototype is capable of sending sweat rate information in real time to either a smartphone or directly to the cloud. Based on a proven theoretical concept, our overall system implementation features a pioneer device that can truly measure the rate of sweat in real time, which was tested and validated on human subjects. Our realization of the real-time sweat rate watch is capable of detecting sweat rates as low as 0.15 µL/min/cm2, with an average error in accuracy of 18% compared to manual sweat rate readings. PMID:29439398

A Real-Time Wireless Sweat Rate Measurement System for Physical Activity Monitoring.

PubMed

Brueck, Andrew; Iftekhar, Tashfin; Stannard, Alicja B; Yelamarthi, Kumar; Kaya, Tolga

2018-02-10

There has been significant research on the physiology of sweat in the past decade, with one of the main interests being the development of a real-time hydration monitor that utilizes sweat. The contents of sweat have been known for decades; sweat provides significant information on the physiological condition of the human body. However, it is important to know the sweat rate as well, as sweat rate alters the concentration of the sweat constituents, and ultimately affects the accuracy of hydration detection. Towards this goal, a calorimetric based flow-rate detection system was built and tested to determine sweat rate in real time. The proposed sweat rate monitoring system has been validated through both controlled lab experiments (syringe pump) and human trials. An Internet of Things (IoT) platform was embedded, with the sensor using a Simblee board and Raspberry Pi. The overall prototype is capable of sending sweat rate information in real time to either a smartphone or directly to the cloud. Based on a proven theoretical concept, our overall system implementation features a pioneer device that can truly measure the rate of sweat in real time, which was tested and validated on human subjects. Our realization of the real-time sweat rate watch is capable of detecting sweat rates as low as 0.15 µL/min/cm², with an average error in accuracy of 18% compared to manual sweat rate readings.

Acting to gain information: Real-time reasoning meets real-time perception

NASA Technical Reports Server (NTRS)

Rosenschein, Stan

1994-01-01

Recent advances in intelligent reactive systems suggest new approaches to the problem of deriving task-relevant information from perceptual systems in real time. The author will describe work in progress aimed at coupling intelligent control mechanisms to real-time perception systems, with special emphasis on frame rate visual measurement systems. A model for integrated reasoning and perception will be discussed, and recent progress in applying these ideas to problems of sensor utilization for efficient recognition and tracking will be described.

Dead-time correction for high-throughput fluorescence lifetime imaging microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Enderlein, Joerg; Ruhlandt, Daja; Chithik, Anna; Ebrecht, René; Wouters, Fred S.; Gregor, Ingo

2016-02-01

Fluorescence lifetime microscopy has become an important method of bioimaging, allowing not only to record intensity and spectral, but also lifetime information across an image. One of the most widely used methods of FLIM is based on Time-Correlated Single Photon Counting (TCSPC). In TCSPC, one determines this curve by exciting molecules with a periodic train of short laser pulses, and then measuring the time delay between the first recorded fluorescence photon after each exciting laser pulse. An important technical detail of TCSPC measurements is the fact that the delay times between excitation laser pulses and resulting fluorescence photons are always measured between a laser pulse and the first fluorescence photon which is detected after that pulse. At high count rates, this leads to so-called pile-up: ``early'' photons eclipse long-delay photons, resulting in heavily skewed TCSPC histograms. To avoid pile-up, a rule of thumb is to perform TCSPC measurements at photon count rates which are at least hundred times smaller than the laser-pulse excitation rate. The downside of this approach is that the fluorescence-photon count-rate is restricted to a value below one hundredth of the laser-pulse excitation-rate, reducing the overall speed with which a fluorescence signal can be measured. We present a new data evaluation method which provides pile-up corrected fluorescence decay estimates from TCSPC measurements at high count rates, and we demonstrate our method on FLIM of fluorescently labeled cells.

Reaction-time-resolved measurements of laser-induced fluorescence in a shock tube with a single laser pulse

NASA Astrophysics Data System (ADS)

Zabeti, S.; Fikri, M.; Schulz, C.

2017-11-01

Shock tubes allow for the study of ultra-fast gas-phase reactions on the microsecond time scale. Because the repetition rate of the experiments is low, it is crucial to gain as much information as possible from each individual measurement. While reaction-time-resolved species concentration and temperature measurements with fast absorption methods are established, conventional laser-induced fluorescence (LIF) measurements with pulsed lasers provide data only at a single reaction time. Therefore, fluorescence methods have rarely been used in shock-tube diagnostics. In this paper, a novel experimental concept is presented that allows reaction-time-resolved LIF measurements with one single laser pulse using a test section that is equipped with several optical ports. After the passage of the shock wave, the reactive mixture is excited along the center of the tube with a 266-nm laser beam directed through a window in the end wall of the shock tube. The emitted LIF signal is collected through elongated sidewall windows and focused onto the entrance slit of an imaging spectrometer coupled to an intensified CCD camera. The one-dimensional spatial resolution of the measurement translates into a reaction-time-resolved measurement while the species information can be gained from the spectral axis of the detected two-dimensional image. Anisole pyrolysis was selected as the benchmark reaction to demonstrate the new apparatus.

Time-resolved fluorescence spectroscopy for chemical sensors

NASA Astrophysics Data System (ADS)

Draxler, Sonja; Lippitsch, Max E.

1996-07-01

A family of sensors is presented with fluorescence decay-time measurements used as the sensing technique. The concept is to take a single fluorophore with a suitably long fluorescence decay time as the basic building block for numerous different sensors. Analyte recognition can be performed by different functional groups that are necessary for selective interaction with the analyte. To achieve this, the principle of excited-state electron transfer is applied with pyrene as the fluorophore. Therefore the same instrumentation based on a small, ambient air-nitrogen laser and solid-state electronics can be used to measure different analytes, for example, oxygen, pH, carbon dioxide, potassium, ammonium, lead, cadmium, zinc, and phosphate.

Real-Time Inhibitor Recession Measurements in Two Space Shuttle Reusable Solid Rocket Motors

NASA Technical Reports Server (NTRS)

McWhorter, B. B.; Ewing, M. E.; Bolton, D. E.; Albrechtsen, K. U.; Earnest, T. E.; Noble, T. C.; Longaker, M.

2003-01-01

Real-time internal motor insulation char line recession measurements have been evaluated for two full-scale static tests of the Space Shuttle Reusable Solid Rocket Motor (RSRM). These char line recession measurements were recorded on the forward facing propellant grain inhibitors to better understand the thermal performance of these inhibitors. The RSRM propellant grain inhibitors are designed to erode away during motor operation, thus making it difficult to use post-fire observations to determine inhibitor thermal performance. Therefore, this new internal motor instrumentation is invaluable in establishing an accurate understanding of inhibitor recession versus motor operation time. The data for the first test was presented at the 37th AIAA/ASME/SAE/ASEE Joint Propulsion Conference and Exhibit (AIAA 2001-3280) in July 2001. Since that time, a second full scale static test has delivered additional real-time data on inhibitor thermal performance. The evaluation of this data is presented in this paper. The second static test, in contrast to the first test, used a slightly different arrangement of instrumentation in the inhibitors. This instrumentation has yielded a better understanding of the inhibitor time dependent inboard tip recession. Graphs of inhibitor recession profiles with time are presented. Inhibitor thermal ablation models have been created from theoretical principals. The model predictions compare favorably with data from both tests. This verified modeling effort is important to support new inhibitor designs for a five segment Space Shuttle solid rocket motor. The internal instrumentation project on RSRM static tests is providing unique opportunities for other real-time internal motor measurements that could not otherwise be directly quantified.

Development and Test of an Infrastructure Free Real-Time Water Level Measurement System

NASA Astrophysics Data System (ADS)

Breuer, E. R.; Heitsenrether, R.; Hensley, W., III; Krug, W.; Wolcott, D.

2016-02-01

NOAA's Center for Operational Oceanographic Products and Services (CO-OPS) is responsible for developing and maintaining the National Water Level Observation Network (NWLON). NWLON consists of over 200 long term observatories that provide near real-time, 6 minute average, water level observations from locations throughout all U.S. coasts. CO-OPS continually analyzes state-of-the-art and emerging technologies to identify potential improvements in data quality and operating efficiency. NOAA, recognizing the changing conditions, anticipates a critical need for real time oceanographic and meteorological observations where traditional approaches are less feasible. CO-OPS is working on the design, development and testing of a real-time tidal measurement system, "The Hermit," for use in coastal regions. The latest prototype has recently completed a successful 3 month field test deployment in the St Andrews Sound region of Georgia, a location where relatively few long term water level records have been collected to date. The test location provided unique challenges such as having a very limited coastal infrastructure and experiencing a 7-8 foot tidal range. The Hermit consists of a bottom mounted pressure/conductivity/temperature sensor (Seabird SBE 26+) and a surface communications buoy which are linked via acoustic modems (Link Quest). The surface buoy relays data back to the CO-OPS database in near-real time using an Iridium satellite based communication system. Additionally, the buoy includes an AirMar all-in-one meteorological sensor. In addition to The Hermit deployment, three test GPS bench marks and a tide staff were installed on a nearby coastline to vertically reference water level measurements. During this deployment, The Hermit successfully provided near real-time measurements of bottom pressure, water conductivity and temperature, wind speed and direction, air temperature, and barometric pressure over the 3 month deployment. During the test period, several

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

PubMed Central

Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

2012-01-01

Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a

Effects of fungal species, cultivation time, growth substrate, and air exposure velocity on the fluorescence properties of airborne fungal spores.

PubMed

Saari, S; Mensah-Attipoe, J; Reponen, T; Veijalainen, A M; Salmela, A; Pasanen, P; Keskinen, J

2015-12-01

Real-time bioaerosol monitoring is possible with fluorescence based instruments. This study provides information on major factors that can affect the fluorescence properties of airborne fungal spores. Two fluorescence-based bioaerosol detectors, BioScout, and ultraviolet aerodynamic particle sizer (UVAPS), were used to study fluorescent particle fractions (FPFs) of released spores of three fungal species (Aspergillus versicolor, Cladosporium cladosporioides, and Penicillium brevicompactum). Two culture media (agar and gypsum board), three ages of the culture (one week, one month, and four months), and three aerosolization air velocities (5, 15, and 27 m/s) were tested. The results showed that the FPF values for spores released from gypsum were typically lower than for those released from agar indicating that poor nutrient substrate produces spores with lower amounts of fluorescent compounds. The results also showed higher FPF values with lower air velocities in aerosolization. This indicates that easily released fully developed spores have more fluorescent compounds compared to forcibly extracted non-matured spores. The FPFs typically were lower with older samples. The FPF results between the two instruments were similar, except with four-month-old samples. The results can be utilized in field measurements of fungal spores to estimate actual concentrations and compare different instruments with fluorescence-based devices as well as in instrument calibration and testing in laboratory conditions. Fluorescence-based instruments are the only choice for real-time detection of fungal spores at the moment. In general, all fluorescence-based bioaerosol instruments are tested against known bacterial and fungal spores in laboratory conditions. This study showed that fungal species, growth substrate, age of culture, and air current exposure rate have an effect on detection efficiency of fungal spores in the fluorescence-based instruments. Therefore, these factors should be

REAL-TIME IN SITU DETECTION OF ORGANIC CONTAMINANTS BY LASER-INDUCED FLUORESCENCE SYSTEM. Final tropical report (Task 1.3).

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

Daniel J. Stepan; James A. Sorensen; Jaroslav Solc

This report summarizes the results of the field demonstration of a laser-induced fluorescence (LIF) method for characterization of brownfields and other contaminated sites. The technology was provided and demonstrated by Dakota Technologies, Inc. (DTI), of Fargo, North Dakota. LIF generates continuous data on the distribution of polycyclic aromatic hydrocarbons (PAHs) within the soil profile. The sensor used to record real-time data is deployed into the soil using a modified truck-mounted Geoprobe percussion soil probing device. The summary of observations described in the following text represents an independent evaluation of the performance, usefulness, and economics of the demonstrated technology for characterizationmore » at PAH-contaminated sites.« less

Measurement of bow tie profiles in CT scanners using a real-time dosimeter

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

Whiting, Bruce R., E-mail: whitingbrucer@gmail.com; Evans, Joshua D.; Williamson, Jeffrey F.

2014-10-15

Purpose: Several areas of computed tomography (CT) research require knowledge about the intensity profile of the x-ray fan beam that is introduced by a bow tie filter. This information is considered proprietary by CT manufacturers, so noninvasive measurement methods are required. One method using real-time dosimeters has been proposed in the literature. A commercially available dosimeter was used to apply that method, and analysis techniques were developed to extract fan beam profiles from measurements. Methods: A real-time ion chamber was placed near the periphery of an empty CT gantry and the dose rate versus time waveform was recorded as themore » x-ray source rotated about the isocenter. In contrast to previously proposed analysis methods that assumed a pointlike detector, the finite-size ion chamber received varying amounts of coverage by the collimated x-ray beam during rotation, precluding a simple relationship between the source intensity as a function of fan beam angle and measured intensity. A two-parameter model for measurement intensity was developed that included both effective collimation width and source-to-detector distance, which then was iteratively solved to minimize the error between duplicate measurements at corresponding fan beam angles, allowing determination of the fan beam profile from measured dose-rate waveforms. Measurements were performed on five different scanner systems while varying parameters such as collimation, kVp, and bow tie filters. On one system, direct measurements of the bow tie profile were collected for comparison with the real-time dosimeter technique. Results: The data analysis method for a finite-size detector was found to produce a fan beam profile estimate with a relative error between duplicate measurement intensities of <5%. It was robust over a wide range of collimation widths (e.g., 1–40 mm), producing fan beam profiles that agreed with a relative error of 1%–5%. Comparison with a direct measurement

A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

PubMed

Han, Yang; Hou, Shao-Yang; Ji, Shang-Zhi; Cheng, Juan; Zhang, Meng-Yue; He, Li-Juan; Ye, Xiang-Zhong; Li, Yi-Min; Zhang, Yi-Xuan

2017-11-15

A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

Real-Time Discrimination and Versatile Profiling of Spontaneous Reactive Oxygen Species in Living Organisms with a Single Fluorescent Probe.

PubMed

Zhang, Ruilong; Zhao, Jun; Han, Guangmei; Liu, Zhengjie; Liu, Cui; Zhang, Cheng; Liu, Bianhua; Jiang, Changlong; Liu, Renyong; Zhao, Tingting; Han, Ming-Yong; Zhang, Zhongping

2016-03-23

Fluorescent probes are powerful tools for the investigations of reactive oxygen species (ROS) in living organisms by visualization and imaging. However, the multiparallel assays of several ROS with multiple probes are often limited by the available number of spectrally nonoverlapping chromophores together with large invasive effects and discrepant biological locations. Meanwhile, the spontaneous ROS profilings in various living organs/tissues are also limited by the penetration capability of probes across different biological barriers and the stability in reactive in vivo environments. Here, we report a single fluorescent probe to achieve the effective discrimination and profiling of hydroxyl radicals (•OH) and hypochlorous acid (HClO) in living organisms. The probe is constructed by chemically grafting an additional five-membered heterocyclic ring and a lateral triethylene glycol chain to a fluorescein mother, which does not only turn off the fluorescence of fluorescein, but also create the dual reactive sites to ROS and the penetration capability in passing through various biological barriers. The reactions of probe with •OH and HClO simultaneously result in cyan and green emissions, respectively, providing the real-time discrimination and quantitative analysis of the two ROS in cellular mitochondria. Surprisingly, the accumulation of probes in the intestine and liver of a normal-state zebrafish and the transfer pathway from intestine-to-blood-to-organ/tissue-to-kidney-to-excretion clearly present the profiling of spontaneous •OH and HClO in these metabolic organs. In particular, the stress generation of •OH at the fresh wound of zebrafish is successfully visualized for the first time, in spite of its extremely short lifetime.

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

PubMed Central

Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

2008-01-01

Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost. PMID:18522756

Using RADFET for the real-time measurement of gamma radiation dose rate

NASA Astrophysics Data System (ADS)

Andjelković, Marko S.; Ristić, Goran S.; Jakšić, Aleksandar B.

2015-02-01

RADFETs (RADiation sensitive Field Effect Transistors) are integrating ionizing radiation dosimeters operating on the principle of conversion of radiation-induced threshold voltage shift into absorbed dose. However, one of the major drawbacks of RADFETs is the inability to provide the information on the dose rate in real-time using the conventional absorbed dose measurement technique. The real-time monitoring of dose rate and absorbed dose can be achieved with the current mode dosimeters such as PN and PIN diodes/photodiodes, but these dosimeters have some limitations as absorbed dose meters and hence they are often not a suitable replacement for RADFETs. In that sense, this paper investigates the possibility of using the RADFET as a real-time dose rate meter so that it could be applied for simultaneous online measurement of the dose rate and absorbed dose. A RADFET sample, manufactured by Tyndall National Institute, Cork, Ireland, was tested as a dose rate meter under gamma irradiation from a Co-60 source. The RADFET was configured as a PN junction, such that the drain, gate and source terminals were grounded, while the radiation-induced current was measured at the bulk terminal, whereby the bulk was successively biased with 0 , 10 , 20  and 30 V. In zero-bias mode the radiation-induced current was unstable, but in the biased mode the current response was stable for the investigated dose rates from 0.65  to 32.1 Gy h-1 and up to the total absorbed dose of 25 Gy. The current increased with the dose rate in accordance with the power law, whereas the sensitivity of the current read-out was linear with respect to the applied bias voltage. Comparison with previously analyzed PIN photodiodes has shown that the investigated RADFET is competitive with PIN photodiodes as a gamma radiation dose rate meter and therefore has the potential to be employed for the real-time monitoring of the dose rate and absorbed dose.

Laser-induced radiation microbeam technology and simultaneous real-time fluorescence imaging in live cells.

PubMed

Botchway, Stanley W; Reynolds, Pamela; Parker, Anthony W; O'Neill, Peter

2012-01-01

The use of nano- and microbeam techniques to induce and identify subcellular localized energy deposition within a region of a living cell provides a means to investigate the effects of low radiation doses. Particularly within the nucleus where the propagation and processing of deoxyribonucleic acid (DNA) damage (and repair) in both targeted and nontargeted cells, the latter being able to study cell-cell (bystander) effects. We have pioneered a near infrared (NIR) femtosecond laser microbeam to mimic ionizing radiation through multiphoton absorption within a 3D femtoliter volume of a highly focused Gaussian laser beam. The novel optical microbeam mimics both complex ionizing and UV-radiation-type cell damage including double strand breaks (DSBs). Using the microbeam technology, we have been able to investigate the formation of DNA DSB and subsequent recruitment of repair proteins to the submicrometer size site of damage introduced in viable cells. The use of a phosphorylated H2AX (γ-H2AX a marker for DSBs, visualized by immunofluorescent staining) and real-time imaging of fluorescently labeling proteins, the dynamics of recruitment of repair proteins in viable mammalian cells can be observed. Here we show the recruitment of ATM, p53 binding protein 1 (53BP1), and RAD51, an integral protein of the homologous recombination process in the DNA repair pathway and Ku-80-GFP involved in the nonhomologous end joining (NHEJ) pathway as exemplar repair process to show differences in the repair kinetics of DNA DSBs. The laser NIR multiphoton microbeam technology shows persistent DSBs at later times post laser irradiation which are indicative of DSBs arising at replication presumably from UV photoproducts or clustered damage containing single strand breaks (SSBs) that are also observed. Effects of the cell cycle may also be investigated in real time. Postirradiation and fixed cells studies show that in G1 cells a fraction of multiphoton laser-induced DSBs is persistent for >6h

Measuring partial fluorescence yield using filtered detectors.

PubMed

Boyko, T D; Green, R J; Moewes, A; Regier, T Z

2014-07-01

Typically, X-ray absorption near-edge structure measurements aim to probe the linear attenuation coefficient. These measurements are often carried out using partial fluorescence yield techniques that rely on detectors having photon energy discrimination improving the sensitivity and the signal-to-background ratio of the measured spectra. However, measuring the partial fluorescence yield in the soft X-ray regime with reasonable efficiency requires solid-state detectors, which have limitations due to the inherent dead-time while measuring. Alternatively, many of the available detectors that are not energy dispersive do not suffer from photon count rate limitations. A filter placed in front of one of these detectors will make the energy-dependent efficiency non-linear, thereby changing the responsivity of the detector. It is shown that using an array of filtered X-ray detectors is a viable method for measuring soft X-ray partial fluorescence yield spectra without dead-time. The feasibility of this technique is further demonstrated using α-Fe2O3 as an example and it is shown that this detector technology could vastly improve the photon collection efficiency at synchrotrons and that these detectors will allow experiments to be completed with a much lower photon flux reducing X-ray-induced damage.

Time-Resolved Measurements in Optoelectronic Microbioanalysis

NASA Technical Reports Server (NTRS)

Bearman, Gregory; Kossakovski, Dmitri

2003-01-01

A report presents discussion of time-resolved measurements in optoelectronic microbioanalysis. Proposed microbioanalytical laboratory-on-a-chip devices for detection of microbes and toxic chemicals would include optoelectronic sensors and associated electronic circuits that would look for fluorescence or phosphorescence signatures of multiple hazardous biomolecules in order to detect which ones were present in a given situation. The emphasis in the instant report is on gating an active-pixel sensor in the time domain, instead of filtering light in the wavelength domain, to prevent the sensor from responding to a laser pulse used to excite fluorescence or phosphorescence while enabling the sensor to respond to the decaying fluorescence or phosphorescence signal that follows the laser pulse. The active-pixel sensor would be turned on after the laser pulse and would be used to either integrate the fluorescence or phosphorescence signal over several lifetimes and many excitation pulses or else take time-resolved measurements of the fluorescence or phosphorescence. The report also discusses issues of multiplexing and of using time-resolved measurements of fluorophores with known different fluorescence lifetimes to distinguish among them.

An orange fluorescent protein tagging system for real-time pollen tracking.

PubMed

Rice, J Hollis; Millwood, Reginald J; Mundell, Richard E; Chambers, Orlando D; Abercrombie, Laura L; Davies, H Maelor; Stewart, C Neal

2013-09-27

Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum × Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification. We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum 'TN 90' and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression. The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.

Real-time direct measurement of liquid (water) evaporation by simple disturbance inhibited interfometry technique

NASA Astrophysics Data System (ADS)

Kim, Yong Gi

2017-11-01

A real-time in-situ interferometry method was proposed to measure water (liquid) evaporation directly over the liquid surface inside the reservoir. The direct evaporation measurement relied on the counting the number of sinusoidal fringes. As the water inside reservoir evaporated, the depth of the water decreases a little thus the optical path length changes. Evaporation signals have been determined as a function of the focusing beam position of the signal beam over the liquid surface. In interferometry technique, the most limiting factors are surface disturbances and vibrations over the liquid surface. This limiting factor was simply inhibited by placing a long cylindrical aluminum tube around the signal beam of the interferometer over the liquid surface. A small diameter cylindrical Al tube diminished vibrations and wind induced surface ripples more effectively than that of the larger one. Water evaporation was successfully measured in real-time with a warm water and cold water even under windy condition with an electric fan. The experimental results demonstrated that the interferometry technique allows determining of liquid evaporation in real-time. Interferometric technique opens up a new possibility of methodology for liquid evaporation measurement even in several environmental disturbances, such as, vibration, surface disturbance, temperature change and windy environments.

Real-Time Systems

DTIC Science & Technology

1992-02-01

Postgraduate School Autonomous Under Vehicle (AUV) are then examined. Autonomous underwater vehicle (AUV), hard real-time system, real - time operating system , real-time programming language, real-time system, soft real-time system.

Binocular Goggle Augmented Imaging and Navigation System provides real-time fluorescence image guidance for tumor resection and sentinel lymph node mapping

PubMed Central

B. Mondal, Suman; Gao, Shengkui; Zhu, Nan; Sudlow, Gail P.; Liang, Kexian; Som, Avik; Akers, Walter J.; Fields, Ryan C.; Margenthaler, Julie; Liang, Rongguang; Gruev, Viktor; Achilefu, Samuel

2015-01-01

The inability to identify microscopic tumors and assess surgical margins in real-time during oncologic surgery leads to incomplete tumor removal, increases the chances of tumor recurrence, and necessitates costly repeat surgery. To overcome these challenges, we have developed a wearable goggle augmented imaging and navigation system (GAINS) that can provide accurate intraoperative visualization of tumors and sentinel lymph nodes in real-time without disrupting normal surgical workflow. GAINS projects both near-infrared fluorescence from tumors and the natural color images of tissue onto a head-mounted display without latency. Aided by tumor-targeted contrast agents, the system detected tumors in subcutaneous and metastatic mouse models with high accuracy (sensitivity = 100%, specificity = 98% ± 5% standard deviation). Human pilot studies in breast cancer and melanoma patients using a near-infrared dye show that the GAINS detected sentinel lymph nodes with 100% sensitivity. Clinical use of the GAINS to guide tumor resection and sentinel lymph node mapping promises to improve surgical outcomes, reduce rates of repeat surgery, and improve the accuracy of cancer staging. PMID:26179014

Binocular Goggle Augmented Imaging and Navigation System provides real-time fluorescence image guidance for tumor resection and sentinel lymph node mapping

NASA Astrophysics Data System (ADS)

B. Mondal, Suman; Gao, Shengkui; Zhu, Nan; Sudlow, Gail P.; Liang, Kexian; Som, Avik; Akers, Walter J.; Fields, Ryan C.; Margenthaler, Julie; Liang, Rongguang; Gruev, Viktor; Achilefu, Samuel

2015-07-01

The inability to identify microscopic tumors and assess surgical margins in real-time during oncologic surgery leads to incomplete tumor removal, increases the chances of tumor recurrence, and necessitates costly repeat surgery. To overcome these challenges, we have developed a wearable goggle augmented imaging and navigation system (GAINS) that can provide accurate intraoperative visualization of tumors and sentinel lymph nodes in real-time without disrupting normal surgical workflow. GAINS projects both near-infrared fluorescence from tumors and the natural color images of tissue onto a head-mounted display without latency. Aided by tumor-targeted contrast agents, the system detected tumors in subcutaneous and metastatic mouse models with high accuracy (sensitivity = 100%, specificity = 98% ± 5% standard deviation). Human pilot studies in breast cancer and melanoma patients using a near-infrared dye show that the GAINS detected sentinel lymph nodes with 100% sensitivity. Clinical use of the GAINS to guide tumor resection and sentinel lymph node mapping promises to improve surgical outcomes, reduce rates of repeat surgery, and improve the accuracy of cancer staging.

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

PubMed

Suin, Vanessa; Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael; Van Gucht, Steven

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

Real-time tricolor phase measuring profilometry based on CCD sensitivity calibration

NASA Astrophysics Data System (ADS)

Zhu, Lin; Cao, Yiping; He, Dawu; Chen, Cheng

2017-02-01

A real-time tricolor phase measuring profilometry (RTPMP) based on charge coupled device (CCD) sensitivity calibration is proposed. Only one colour fringe pattern whose red (R), green (G) and blue (B) components are, respectively, coded as three sinusoidal phase-shifting gratings with an equivalent shifting phase of 2π/3 is needed and sent to an appointed flash memory on a specialized digital light projector (SDLP). A specialized time-division multiplexing timing sequence actively controls the SDLP to project the fringe patterns in R, G and B channels sequentially onto the measured object in one over seventy-two of a second and meanwhile actively controls a high frame rate monochrome CCD camera to capture the corresponding deformed patterns synchronously with the SDLP. So the sufficient information for reconstructing the three-dimensional (3D) shape in one over twenty-four of a second is obtained. Due to the different spectral sensitivity of the CCD camera to RGB lights, the captured deformed patterns from R, G and B channels cannot share the same peak and valley, which will lead to lower accuracy or even failing to reconstruct the 3D shape. So a deformed pattern amending method based on CCD sensitivity calibration is developed to guarantee the accurate 3D reconstruction. The experimental results verify the feasibility of the proposed RTPMP method. The proposed RTPMP method can obtain the 3D shape at over the video frame rate of 24 frames per second, avoid the colour crosstalk completely and be effective for measuring real-time changing object.

Detection of Explosive Vapors: The Roles of Exciton and Molecular Diffusion in Real-Time Sensing.

PubMed

Ali, Mohammad A; Shoaee, Safa; Fan, Shengqiang; Burn, Paul L; Gentle, Ian R; Meredith, Paul; Shaw, Paul E

2016-11-04

Time-resolved quartz crystal microbalance with in situ fluorescence measurements are used to monitor the sorption of the nitroaromatic (explosive) vapor, 2,4-dinitrotoluene (DNT) into a porous pentiptycene-containing poly(phenyleneethynylene) sensing film. Correlation of the nitroaromatic mass uptake with fluorescence quenching shows that the analyte diffusion follows the Case-II transport model, a film-swelling-limited process, in which a sharp diffusional front propagates at a constant velocity through the film. At a low vapor pressure of DNT of ≈16 ppb, the analyte concentration in the front is sufficiently high to give an average fluorophore-analyte separation of ≈1.5 nm. Hence, a long exciton diffusion length is not required for real-time sensing in the solid state. Rather the diffusion behavior of the analyte and the strength of the binding interaction between the analyte and the polymer play first-order roles in the fluorescence quenching process. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using Indirect Turbulence Measurements for Real-Time Parameter Estimation in Turbulent Air

NASA Technical Reports Server (NTRS)

Martos, Borja; Morelli, Eugene A.

2012-01-01

The use of indirect turbulence measurements for real-time estimation of parameters in a linear longitudinal dynamics model in atmospheric turbulence was studied. It is shown that measuring the atmospheric turbulence makes it possible to treat the turbulence as a measured explanatory variable in the parameter estimation problem. Commercial off-the-shelf sensors were researched and evaluated, then compared to air data booms. Sources of colored noise in the explanatory variables resulting from typical turbulence measurement techniques were identified and studied. A major source of colored noise in the explanatory variables was identified as frequency dependent upwash and time delay. The resulting upwash and time delay corrections were analyzed and compared to previous time shift dynamic modeling research. Simulation data as well as flight test data in atmospheric turbulence were used to verify the time delay behavior. Recommendations are given for follow on flight research and instrumentation.

Shelf Life of Food Products: From Open Labeling to Real-Time Measurements.

PubMed

Corradini, Maria G

2018-03-25

The labels currently used on food and beverage products only provide consumers with a rough guide to their expected shelf lives because they assume that a product only experiences a limited range of predefined handling and storage conditions. These static labels do not take into consideration conditions that might shorten a product's shelf life (such as temperature abuse), which can lead to problems associated with food safety and waste. Advances in shelf-life estimation have the potential to improve the safety, reliability, and sustainability of the food supply. Selection of appropriate kinetic models and data-analysis techniques is essential to predict shelf life, to account for variability in environmental conditions, and to allow real-time monitoring. Novel analytical tools to determine safety and quality attributes in situ coupled with modern tracking technologies and appropriate predictive tools have the potential to provide accurate estimations of the remaining shelf life of a food product in real time. This review summarizes the necessary steps to attain a transition from open labeling to real-time shelf-life measurements.

In situ real-time measurement of physical characteristics of airborne bacterial particles

NASA Astrophysics Data System (ADS)

Jung, Jae Hee; Lee, Jung Eun

2013-12-01

Bioaerosols, including aerosolized bacteria, viruses, and fungi, are associated with public health and environmental problems. One promising control method to reduce the harmful effects of bioaerosols is thermal inactivation via a continuous-flow high-temperature short-time (HTST) system. However, variations in bioaerosol physical characteristics - for example, the particle size and shape - during the continuous-flow inactivation process can change the transport properties in the air, which can affect particle deposition in the human respiratory system or the filtration efficiency of ventilation systems. Real-time particle monitoring techniques are a desirable alternative to the time-consuming process of microscopic analysis that is conventionally used in sampling and particle characterization. Here, we report in situ real-time optical scattering measurements of the physical characteristics of airborne bacteria particles following an HTST process in a continuous-flow system. Our results demonstrate that the aerodynamic diameter of bacterial aerosols decreases when exposed to a high-temperature environment, and that the shape of the bacterial cells is significantly altered. These variations in physical characteristics using optical scattering measurements were found to be in agreement with the results of scanning electron microscopy analysis.

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

Grayscale imbalance correction in real-time phase measuring profilometry

NASA Astrophysics Data System (ADS)

Zhu, Lin; Cao, Yiping; He, Dawu; Chen, Cheng

2016-10-01

Grayscale imbalance correction in real-time phase measuring profilometry (RPMP) is proposed. In the RPMP, the sufficient information is obtained to reconstruct the 3D shape of the measured object in one over twenty-four of a second. Only one color fringe pattern whose R, G and B channels are coded as three sinusoidal phase-shifting gratings with an equivalent shifting phase of 2π/3 is sent to a flash memory on a specialized digital light projector (SDLP). And then the SDLP projects the fringe patterns in R, G and B channels sequentially onto the measured object in one over seventy-two of a second and meanwhile a monochrome CCD camera captures the corresponding deformed patterns synchronously with the SDLP. Because the deformed patterns from three color channels are captured at different time, the color crosstalk is avoided completely. But due to the monochrome CCD camera's different spectral sensitivity to R, G and B tricolor, there will be grayscale imbalance among these deformed patterns captured at R, G and B channels respectively which may result in increasing measuring errors or even failing to reconstruct the 3D shape. So a new grayscale imbalance correction method based on least square method is developed. The experimental results verify the feasibility of the proposed method.

Thermal damage assessment of blood vessels in a hamster skin flap model by fluorescence measurement of a liposome-dye system.

PubMed

Mordon, S; Desmettre, T; Devoisselle, J M; Soulie, S

1997-01-01

The present study was undertaken to evaluate the feasibility of thermal damage assessment of blood vessels by using laser-induced release of liposome-encapsulated dye. Experiments were performed in a hamster skin flap model. Laser irradiation was achieved with a 300 microm fiber connected to a 805 nm diode laser (power = 0.8W, spot diameter = 1.3 mm and pulse exposure time lasting from 1 to 6 s) after potentiation using a specific indocyanine green (ICG) formulation (water and oil emulsion). Liposomes-encapsulated carboxyfluorescein were prepared by the sonication procedure. Carboxyfluorescein (5,6-CF) was loaded at high concentration (100 mM) in order to quench its fluorescence. The measurements were performed after i.v. injection of DSPC liposomes (1.5 ml) and lasted 40 min. Fluorescence emission was measured with an ultra high sensitivity intensified camera. Three different shapes of fluorescent spots were identified depending on target (blood vessel or skin) and energy deposition in tissue: (i) intravascular fluorescence, (ii) transient low fluorescence circular spot, and (iii) persistent high intense fluorescence spot. These images are correlated with histological data. Real-time fluorescence imaging seems to be a good tool to estimate in a non-invasive manner the thermal damage induced by a diode laser combined with ICG potentiation.

Discriminating Bio-aerosols from Non-Bio-aerosols in Real-Time by Pump-Probe Spectroscopy

PubMed Central

Sousa, Gustavo; Gaulier, Geoffrey; Bonacina, Luigi; Wolf, Jean-Pierre

2016-01-01

The optical identification of bioaerosols in the atmosphere and its discrimination against combustion related particles is a major issue for real-time, field compatible instruments. In the present paper, we show that by embedding advanced pump-probe depletion spectroscopy schemes in a portable instrument, it is possible to discriminate amino acid containing airborne particles (bacteria, humic particles, etc.) from poly-cyclic aromatic hydrocarbon containing combustion particles (Diesel droplets, soot, vehicle exhausts) with high selectivity. Our real-time, multi-modal device provides, in addition to the pump-probe depletion information, fluorescence spectra (over 32 channels), fluorescence lifetime and Mie scattering patterns of each individually flowing particle in the probed air. PMID:27619546

Discriminating Bio-aerosols from Non-Bio-aerosols in Real-Time by Pump-Probe Spectroscopy

NASA Astrophysics Data System (ADS)

Sousa, Gustavo; Gaulier, Geoffrey; Bonacina, Luigi; Wolf, Jean-Pierre

2016-09-01

The optical identification of bioaerosols in the atmosphere and its discrimination against combustion related particles is a major issue for real-time, field compatible instruments. In the present paper, we show that by embedding advanced pump-probe depletion spectroscopy schemes in a portable instrument, it is possible to discriminate amino acid containing airborne particles (bacteria, humic particles, etc.) from poly-cyclic aromatic hydrocarbon containing combustion particles (Diesel droplets, soot, vehicle exhausts) with high selectivity. Our real-time, multi-modal device provides, in addition to the pump-probe depletion information, fluorescence spectra (over 32 channels), fluorescence lifetime and Mie scattering patterns of each individually flowing particle in the probed air.

Discriminating Bio-aerosols from Non-Bio-aerosols in Real-Time by Pump-Probe Spectroscopy.

PubMed

Sousa, Gustavo; Gaulier, Geoffrey; Bonacina, Luigi; Wolf, Jean-Pierre

2016-09-13

The optical identification of bioaerosols in the atmosphere and its discrimination against combustion related particles is a major issue for real-time, field compatible instruments. In the present paper, we show that by embedding advanced pump-probe depletion spectroscopy schemes in a portable instrument, it is possible to discriminate amino acid containing airborne particles (bacteria, humic particles, etc.) from poly-cyclic aromatic hydrocarbon containing combustion particles (Diesel droplets, soot, vehicle exhausts) with high selectivity. Our real-time, multi-modal device provides, in addition to the pump-probe depletion information, fluorescence spectra (over 32 channels), fluorescence lifetime and Mie scattering patterns of each individually flowing particle in the probed air.

Monitoring airborne molecular contamination: a quantitative and qualitative comparison of real-time and grab-sampling techniques

NASA Astrophysics Data System (ADS)

Shupp, Aaron M.; Rodier, Dan; Rowley, Steven

2007-03-01

Monitoring and controlling Airborne Molecular Contamination (AMC) has become essential in deep ultraviolet (DUV) photolithography for both optimizing yields and protecting tool optics. A variety of technologies have been employed for both real-time and grab-sample monitoring. Real-time monitoring has the advantage of quickly identifying "spikes" and upset conditions, while 2 - 24 hour plus grab sampling allows for extremely low detection limits by concentrating the mass of the target contaminant over a period of time. Employing a combination of both monitoring techniques affords the highest degree of control, lowest detection limits, and the most detailed data possible in terms of speciation. As happens with many technologies, there can be concern regarding the accuracy and agreement between real-time and grab-sample methods. This study utilizes side by side comparisons of two different real-time monitors operating in parallel with both liquid impingers and dry sorbent tubes to measure NIST traceable gas standards as well as real world samples. By measuring in parallel, a truly valid comparison is made between methods while verifying the results against a certified standard. The final outcome for this investigation is that a dry sorbent tube grab-sample technique produced results that agreed in terms of accuracy with NIST traceable standards as well as the two real-time techniques Ion Mobility Spectrometry (IMS) and Pulsed Fluorescence Detection (PFD) while a traditional liquid impinger technique showed discrepancies.

Real-time, intraoperative detection of residual breast cancer in lumpectomy cavity walls using a novel cathepsin-activated fluorescent imaging system.

PubMed

Smith, Barbara L; Gadd, Michele A; Lanahan, Conor R; Rai, Upahvan; Tang, Rong; Rice-Stitt, Travis; Merrill, Andrea L; Strasfeld, David B; Ferrer, Jorge M; Brachtel, Elena F; Specht, Michelle C

2018-06-09

Obtaining tumor-free surgical margins is critical to prevent recurrence in breast-conserving surgery but it remains challenging. We assessed the LUM Imaging System for real-time, intraoperative detection of residual tumor. Lumpectomy cavity walls and excised specimens of breast cancer lumpectomy patients were assessed with the LUM Imaging System (Lumicell, Inc., Wellesley MA) with and without intravenous LUM015, a cathepsin-activatable fluorescent agent. Fluorescence at potential sites of residual tumor was evaluated with a sterile hand-held probe, displayed on a monitor and correlated with histopathology. Background autofluorescence was assessed in excised specimens from 9 patients who did not receive LUM015. In vivo lumpectomy cavities and excised specimens were then imaged in 15 women undergoing breast cancer surgery who received no LUM015, 0.5, or 1 mg/kg LUM015 (5 women per dose). Among these, 11 patients had invasive carcinoma with ductal carcinoma in situ (DCIS) and 4 had only DCIS. Image acquisition took 1 s for each 2.6-cm-diameter surface. No significant background normal breast fluorescence was identified. Elevated fluorescent signal was seen from invasive cancers and DCIS. Mean tumor-to-normal signal ratios were 4.70 ± 1.23 at 0.5 mg/kg and 4.22 ± 0.9 at 1.0 mg/kg (p = 0.54). Tumor was distinguished from normal tissue in pre-and postmenopausal women and readings were not affected by breast density. Some benign tissues produced fluorescent signal with LUM015. The LUM Imaging System allows rapid identification of residual tumor in the lumpectomy cavity of breast cancer patients and may reduce rates of positive margins.

Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

NASA Astrophysics Data System (ADS)

Zhang, Yingpei; Haskins, Catherine; Lopez-Cruzan, Marisa; Zhang, Jianhua; Centonze, Victoria E.; Herman, Brian

2004-08-01

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP caspase 3 substrate YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

Real-time measurement and monitoring of absorbed dose for electron beams

NASA Astrophysics Data System (ADS)

Korenev, Sergey; Korenev, Ivan; Rumega, Stanislav; Grossman, Leon

2004-09-01

The real-time method and system for measurement and monitoring of absorbed dose for industrial and research electron accelerators is considered in the report. The system was created on the basis of beam parameters method. The main concept of this method consists in the measurement of dissipated kinetic energy of electrons in the irradiated product, determination of number of electrons and mass of irradiated product in the same cell by following calculation of absorbed dose in the cell. The manual and automation systems for dose measurements are described. The systems are acceptable for all types of electron accelerators.

Real-time thickness measurement of MCC ablator material

NASA Technical Reports Server (NTRS)

Greenway, R. Bryan, Jr.

1994-01-01

One of the most favorable characteristics of the Space Shuttle Program is the reusability of two of its primary components: the orbiter itself and the Solid Rocket Boosters (SRB). The SRB's provide the primary source of propulsion for the Space Shuttle during take-off after which they are recovered for refurbishment and reuse. During refurbishment, the SRB's are stripped of all remaining ablative (heat resistant) coating. A new layer is applied to the appropriate sections (nose cone, frustum, forward skirt, and aft skirt). It is the process of applying the ablative coating which provided the impetus for this project. The thickness of this protective layer is considered to be of primary importance to the level of thermal protection provided. The objectives of this effort are to investigate possible techniques for measuring the thickness of MCC, and if possible to test the specific capabilities of those considered good candidates for implementation. The system would be able to take measurements in real-time as close to the spray gun as possible. This will allow the information to be used in the control of the process without an inordinate time delay between a measurement and its appropriate response. The thickness of the deposited material is to be measured with less than 0.100 in if uncertainty. This is the defined tolerance window for the ablator thickness. Finally, it must operate within the confines of the chamber which encloses the turntable, robot, and spray system, and therefore is required to be insensitive to, or at least maintainable in, that environment.

A real-time measurement system for long-life flood monitoring and warning applications.

PubMed

Marin-Perez, Rafael; García-Pintado, Javier; Gómez, Antonio Skarmeta

2012-01-01

A flood warning system incorporates telemetered rainfall and flow/water level data measured at various locations in the catchment area. Real-time accurate data collection is required for this use, and sensor networks improve the system capabilities. However, existing sensor nodes struggle to satisfy the hydrological requirements in terms of autonomy, sensor hardware compatibility, reliability and long-range communication. We describe the design and development of a real-time measurement system for flood monitoring, and its deployment in a flash-flood prone 650 km(2) semiarid watershed in Southern Spain. A developed low-power and long-range communication device, so-called DatalogV1, provides automatic data gathering and reliable transmission. DatalogV1 incorporates self-monitoring for adapting measurement schedules for consumption management and to capture events of interest. Two tests are used to assess the success of the development. The results show an autonomous and robust monitoring system for long-term collection of water level data in many sparse locations during flood events.

A Real-Time Measurement System for Long-Life Flood Monitoring and Warning Applications

PubMed Central

Marin-Perez, Rafael; García-Pintado, Javier; Gómez, Antonio Skarmeta

2012-01-01

A flood warning system incorporates telemetered rainfall and flow/water level data measured at various locations in the catchment area. Real-time accurate data collection is required for this use, and sensor networks improve the system capabilities. However, existing sensor nodes struggle to satisfy the hydrological requirements in terms of autonomy, sensor hardware compatibility, reliability and long-range communication. We describe the design and development of a real-time measurement system for flood monitoring, and its deployment in a flash-flood prone 650 km2 semiarid watershed in Southern Spain. A developed low-power and long-range communication device, so-called DatalogV1, provides automatic data gathering and reliable transmission. DatalogV1 incorporates self-monitoring for adapting measurement schedules for consumption management and to capture events of interest. Two tests are used to assess the success of the development. The results show an autonomous and robust monitoring system for long-term collection of water level data in many sparse locations during flood events. PMID:22666028

Real-time digital heterodyne interferometer for high resolution plasma density measurements at ISTTOK.

PubMed

Marques, T G; Gouveia, A; Pereira, T; Fortunato, J; Carvalho, B B; Sousa, J; Silva, C; Fernandes, H

2008-10-01

With the implementation of alternating discharges (ac) at the ISTTOK tokamak, the typical duration of the discharges increased from 35 to 250 ms. This time increase created the need for a real-time electron density measurement in order to control the plasma fueling. The diagnostic chosen for the real-time calculation was the microwave interferometer. The ISTTOK microwave interferometer is a heterodyne system with quadrature detection and a probing frequency of 100 GHz (lambda(0)=3 mm). In this paper, a low-cost approach for real-time diagnostic using a digital signal programmable intelligent computer embedded system is presented, which allows the measurement of the phase with a 1% fringe accuracy in less than 6 micros. The system increases its accuracy by digitally correcting the offsets of the input signals and making use of a judicious lookup table optimized to improve the nonlinear behavior of the transfer curve. The electron density is determined at a rate of 82 kHz (limited by the analog to digital converter), and the data are transmitted for each millisecond although this last parameter could be much lower (around 12 micros--each value calculated is transmitted). In the future, this same system is expected to control plasma actuators, such as the piezoelectric valve of the hydrogen injection system responsible for the plasma fueling.

Real-time heart rate measurement for multi-people using compressive tracking

NASA Astrophysics Data System (ADS)

Liu, Lingling; Zhao, Yuejin; Liu, Ming; Kong, Lingqin; Dong, Liquan; Ma, Feilong; Pang, Zongguang; Cai, Zhi; Zhang, Yachu; Hua, Peng; Yuan, Ruifeng

2017-09-01

The rise of aging population has created a demand for inexpensive, unobtrusive, automated health care solutions. Image PhotoPlethysmoGraphy(IPPG) aids in the development of these solutions by allowing for the extraction of physiological signals from video data. However, the main deficiencies of the recent IPPG methods are non-automated, non-real-time and susceptible to motion artifacts(MA). In this paper, a real-time heart rate(HR) detection method for multiple subjects simultaneously was proposed and realized using the open computer vision(openCV) library, which consists of getting multiple subjects' facial video automatically through a Webcam, detecting the region of interest (ROI) in the video, reducing the false detection rate by our improved Adaboost algorithm, reducing the MA by our improved compress tracking(CT) algorithm, wavelet noise-suppression algorithm for denoising and multi-threads for higher detection speed. For comparison, HR was measured simultaneously using a medical pulse oximetry device for every subject during all sessions. Experimental results on a data set of 30 subjects show that the max average absolute error of heart rate estimation is less than 8 beats per minute (BPM), and the processing speed of every frame has almost reached real-time: the experiments with video recordings of ten subjects under the condition of the pixel resolution of 600× 800 pixels show that the average HR detection time of 10 subjects was about 17 frames per second (fps).

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

PubMed Central

Fruhwirth, Gilbert O.; Ameer-Beg, Simon; Cook, Richard; Watson, Timothy; Ng, Tony; Festy, Frederic

2010-01-01

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications. PMID:20588974

Development of defined microbial population standards using fluorescence activated cell sorting for the absolute quantification of S. aureus using real-time PCR.

PubMed

Martinon, Alice; Cronin, Ultan P; Wilkinson, Martin G

2012-01-01

In this article, four types of standards were assessed in a SYBR Green-based real-time PCR procedure for the quantification of Staphylococcus aureus (S. aureus) in DNA samples. The standards were purified S. aureus genomic DNA (type A), circular plasmid DNA containing a thermonuclease (nuc) gene fragment (type B), DNA extracted from defined populations of S. aureus cells generated by Fluorescence Activated Cell Sorting (FACS) technology with (type C) or without purification of DNA by boiling (type D). The optimal efficiency of 2.016 was obtained on Roche LightCycler(®) 4.1. software for type C standards, whereas the lowest efficiency (1.682) corresponded to type D standards. Type C standards appeared to be more suitable for quantitative real-time PCR because of the use of defined populations for construction of standard curves. Overall, Fieller Confidence Interval algorithm may be improved for replicates having a low standard deviation in Cycle Threshold values such as found for type B and C standards. Stabilities of diluted PCR standards stored at -20°C were compared after 0, 7, 14 and 30 days and were lower for type A or C standards compared with type B standards. However, FACS generated standards may be useful for bacterial quantification in real-time PCR assays once optimal storage and temperature conditions are defined.

Characterization of time-resolved fluorescence response measurements for distributed optical-fiber sensing.

PubMed

Sinchenko, Elena; Gibbs, W E Keith; Davis, Claire E; Stoddart, Paul R

2010-11-20

A distributed optical-fiber sensing system based on pulsed excitation and time-gated photon counting has been used to locate a fluorescent region along the fiber. The complex Alq3 and the infrared dye IR-125 were examined with 405 and 780 nm excitation, respectively. A model to characterize the response of the distributed fluorescence sensor to a Gaussian input pulse was developed and tested. Analysis of the Alq3 fluorescent response confirmed the validity of the model and enabled the fluorescence lifetime to be determined. The intrinsic lifetime obtained (18.2±0.9 ns) is in good agreement with published data. The decay rate was found to be proportional to concentration, which is indicative of collisional deactivation. The model allows the spatial resolution of a distributed sensing system to be improved for fluorophores with lifetimes that are longer than the resolution of the sensing system.

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

PubMed Central

Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R.; Smith, T. F.

2006-01-01

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. PMID:16418529

Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement.

PubMed

Renault, Thibaud T; Luna-Vargas, Mark P A; Chipuk, Jerry E

2016-08-05

The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al ., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is "Liver mitochondria isolation" in which the liver is dissected from mice to obtain mitochondria. "Mitochondria labeling with JC-1 and size fractionation" is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the "Real-time MOMP measurements" protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al ., 2015).

A laser scanning confocal imaging-surface plasmon resonance system application in real time detection of antibody-antigen interaction

NASA Astrophysics Data System (ADS)

Zhang, H. Y.; Yang, L. Q.; Liu, W. M.

2011-12-01

The laser scanning confocal microscope (LSCM) offers several advantages over conventional optical microscopy, but most LSCM work is qualitative analysis and it is very hard to achieve quantitative detection directly with the changing of the fluorescent intensity. A new real time sensor system for the antibody-antigen interaction detection was built integrating with a LSCM and a wavelength-dependent surface plasmon resonance (SPR) sensor. The system was applied to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibody in real time. The fluorescence images changing is well with that of SPR wavelengths in real time, and the trend of the resonance wavelength shift with the concentrations of antibody is similar to that of the fluorescent intensity changing. The results show that SPR makes up the short of quantificational analysis with LSCM with the high spatial resolution. The sensor system shows the merits of the of the LSCM and SPR synergetic application, which are of great importance for practical application in biosensor and life science for interesting local interaction.

Single frequency GPS measurements in real-time artificial satellite orbit determination

NASA Astrophysics Data System (ADS)

Chiaradia, orbit determination A. P. M.; Kuga, H. K.; Prado, A. F. B. A.

2003-07-01

A simplified and compact algorithm with low computational cost providing an accuracy around tens of meters for artificial satellite orbit determination in real-time and on-board is developed in this work. The state estimation method is the extended Kalman filter. The Cowell's method is used to propagate the state vector, through a simple Runge-Kutta numerical integrator of fourth order with fixed step size. The modeled forces are due to the geopotential up to 50th order and degree of JGM-2 model. To time-update the state error covariance matrix, it is considered a simplified force model. In other words, in computing the state transition matrix, the effect of J 2 (Earth flattening) is analytically considered, which unloads dramatically the processing time. In the measurement model, the single frequency GPS pseudorange is used, considering the effects of the ionospheric delay, clock offsets of the GPS and user satellites, and relativistic effects. To validate this model, real live data are used from Topex/Poseidon satellite and the results are compared with the Topex/Poseidon Precision Orbit Ephemeris (POE) generated by NASA/JPL, for several test cases. It is concluded that this compact algorithm enables accuracies of tens of meters with such simplified force model, analytical approach for computing the transition matrix, and a cheap GPS receiver providing single frequency pseudorange measurements.

Portable, real-time alloy identification of metallic wear debris from machinery lubrication systems: laser-induced breakdown spectroscopy versus x-ray fluorescence

NASA Astrophysics Data System (ADS)

Suresh, Pooja

2014-05-01

Alloy identification of oil-borne wear debris captured on chip detectors, filters and magnetic plugs allows the machinery maintainer to assess the health of the engine or gearbox and identify specific component damage. Today, such identification can be achieved in real time using portable, at-line laser-induced breakdown spectroscopy (LIBS) and Xray fluorescence (XRF) instruments. Both techniques can be utilized in various industries including aviation, marine, railways, heavy diesel and other industrial machinery with, however, some substantial differences in application and instrument performance. In this work, the performances of a LIBS and an XRF instrument are compared based on measurements of a wide range of typical aerospace alloys including steels, titanium, aluminum and nickel alloys. Measurement results were analyzed with a staged correlation technique specifically developed for the purposes of this study - identifying the particle alloy composition using a pre-recorded library of spectral signatures. The analysis is performed in two stages: first, the base element of the alloy is determined by correlation with the stored elemental spectra and then, the alloy is identified by matching the particle's spectral signature using parametric correlation against the stored spectra of all alloys that have the same base element. The correlation analysis has achieved highly repeatable discrimination between alloys of similar composition. Portable LIBS demonstrates higher detection accuracy and better identification of alloys comprising lighter elements as compared to that of the portable XRF system, and reveals a significant reduction in the analysis time over XRF.

Excitation-emission matrices measurements of human cutaneous lesions: tool for fluorescence origin

NASA Astrophysics Data System (ADS)

Zhelyazkova, A.; Borisova, E.; Angelova, L.; Pavlova, E.; Keremedchiev, M.

2013-11-01

The light induced fluorescence (LIF) technique has the potential of providing real-time diagnosis of malignant and premalignant skin tissue; however, human skin is a multilayered and inhomogeneous organ with different optical properties that complicate the analysis of cutaneous fluorescence spectra. In spite of the difficulties related to the detection and analysis of fluorescent data from skin lesions, this technique is among the most widely applied techniques in laboratorial and pre-clinical investigations for early skin neoplasia diagnosis. The important point is to evaluate all sources of intrinsic fluorescence and find any significant alterations distinguishing the normal skin from a cancerous state of the tissue; this would make the autofluorescence signal obtained useful for the development of a non-invasive diagnostic tool for the dermatological practice. Our investigations presented here were based on ex vivo point-by-point measurements of excitation-emission matrices (EEM) from excised tumor lesions and the surrounding skin taken during the daily clinical practice of Queen Jiovanna- ISUL University Hospital, Sofia, the local Ethical Committee's approval having already been obtained. The fluorescence emission was measured between 300 nm and 800 nm using excitation in the 280-440 nm spectral range. In the process of excitation-emission matrices (EEM) measurements we could establish the origin of the autofluorescence and the compounds related by assigning the excitation and emission maxima obtained during the experiments. The EEM were compared for normal human skin, basal cell carcinoma, squamous cell carcinoma, benign nevi and malignant melanoma lesions to obtain information for the most common skin malignancies and their precursors. The main spectral features and the applicability of the technique of autofluorescent spectroscopy of human skin in general as an initial diagnostic tool are discussed as well.

Real-time depth measurement for micro-holes drilled by lasers

NASA Astrophysics Data System (ADS)

Lin, Cheng-Hsiang; Powell, Rock A.; Jiang, Lan; Xiao, Hai; Chen, Shean-Jen; Tsai, Hai-Lung

2010-02-01

An optical system based on the confocal principle has been developed for real-time precision measurements of the depth of micro-holes during the laser drilling process. The capability of the measuring system is theoretically predicted by the Gaussian lens formula and experimentally validated to achieve a sensitivity of 0.5 µm. A nanosecond laser system was used to drill holes, and the hole depths were measured by the proposed measuring system and by the cut-and-polish method. The differences between these two measurements are found to be 5.0% for hole depths on the order of tens of microns and 11.2% for hundreds of microns. The discrepancies are caused mainly by the roughness of the bottom surface of the hole and by the existence of debris in the hole. This system can be easily implemented in a laser workstation for the fabrication of 3D microstructures.

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

PubMed

Ponchel, Frederique; Toomes, Carmel; Bransfield, Kieran; Leong, Fong T; Douglas, Susan H; Field, Sarah L; Bell, Sandra M; Combaret, Valerie; Puisieux, Alain; Mighell, Alan J; Robinson, Philip A; Inglehearn, Chris F; Isaacs, John D; Markham, Alex F

2003-10-13

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

PubMed

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

Real-time high dynamic range laser scanning microscopy

NASA Astrophysics Data System (ADS)

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-04-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice.

PubMed

Oliveira, Janaina Correia; da Silva, Aline Caroline; Oliveira, Renato Antonio Dos Santos; Pereira, Valéria Rêgo Alves; Gil, Laura Helena Vega Gonzales

2016-11-01

The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis. Copyright © 2016 Elsevier B.V. All rights reserved.

Blending DNA binding dyes to improve detection in real-time PCR.

PubMed

Jansson, Linda; Koliana, Marianne; Sidstedt, Maja; Hedman, Johannes

2017-03-01

The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.

Real-time Fluorescence Image-Guided Oncologic Surgery

PubMed Central

Mondal, Suman B.; Gao, Shengkui; Zhu, Nan; Liang, Rongguang; Gruev, Viktor; Achilefu, Samuel

2014-01-01

Medical imaging plays a critical role in cancer diagnosis and planning. Many of these patients rely on surgical intervention for curative outcomes. This requires a careful identification of the primary and microscopic tumors, and the complete removal of cancer. Although there have been efforts to adapt traditional imaging modalities for intraoperative image guidance, they suffer from several constraints such as large hardware footprint, high operation cost, and disruption of the surgical workflow. Because of the ease of image acquisition, relatively low cost devices and intuitive operation, optical imaging methods have received tremendous interests for use in real-time image-guided surgery. To improve imaging depth under low interference by tissue autofluorescence, many of these applications utilize light in the near-infra red (NIR) wavelengths, which is invisible to human eyes. With the availability of a wide selection of tumor-avid contrast agents, advancements in imaging sensors, electronic and optical designs, surgeons are able to combine different attributes of NIR optical imaging techniques to improve treatment outcomes. The emergence of diverse commercial and experimental image guidance systems, which are in various stages of clinical translation, attests to the potential high impact of intraoperative optical imaging methods to improve speed of oncologic surgery with high accuracy and minimal margin positivity. PMID:25287689

Environmental Measurement-While-Drilling system for real-time field screening of contaminants

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

Lockwood, G.J.; Normann, R.A.; Bishop, L.B.

Sampling during environmental drilling is essential to fully characterize the spatial distribution and migration of near surface contaminants. However, the analysis of these samples is not only expensive, but can take weeks or months when sent to an off-site laboratory. In contrast, measurement-while-drilling (MWD) screening capability could save money and valuable time by quickly distinguishing between contaminated and uncontaminated areas. Real-time measurements provided by a MVM system would enable on-the-spot decisions to be made regarding sampling strategies, enhance worker safety, and provide the added flexibility of being able to ``steer`` the drill bit in or out hazardous zones. During measurement-while-drilling,more » down-hole sensors are located behind the drill bit and linked by a rapid data transmission system to a computer at the surface. As drilling proceeds, data are collected on the nature and extent of the subsurface contamination in real-time. The down-hole sensor is a Geiger-Mueller tube (GMT) gamma radiation detector. In addition to the GMT signal, the MWD system monitors these required down-hole voltages and two temperatures associated with the detector assembly. The Gamma Ray Detection System (GRDS) and electronics package are discussed in as well as the results of the field test. Finally, our conclusions and discussion of future work are presented.« less

Real-time high-resolution heterodyne-based measurements of spectral dynamics in fibre lasers

PubMed Central

Sugavanam, Srikanth; Fabbri, Simon; Le, Son Thai; Lobach, Ivan; Kablukov, Sergey; Khorev, Serge; Churkin, Dmitry

2016-01-01

Conventional tools for measurement of laser spectra (e.g. optical spectrum analysers) capture data averaged over a considerable time period. However, the generation spectrum of many laser types may involve spectral dynamics whose relatively fast time scale is determined by their cavity round trip period, calling for instrumentation featuring both high temporal and spectral resolution. Such real-time spectral characterisation becomes particularly challenging if the laser pulses are long, or they have continuous or quasi-continuous wave radiation components. Here we combine optical heterodyning with a technique of spatio-temporal intensity measurements that allows the characterisation of such complex sources. Fast, round-trip-resolved spectral dynamics of cavity-based systems in real-time are obtained, with temporal resolution of one cavity round trip and frequency resolution defined by its inverse (85 ns and 24 MHz respectively are demonstrated). We also show how under certain conditions for quasi-continuous wave sources, the spectral resolution could be further increased by a factor of 100 by direct extraction of phase information from the heterodyned dynamics or by using double time scales within the spectrogram approach. PMID:26984634

Adapting CALIPSO Climate Measurements for Near Real Time Analyses and Forecasting

NASA Technical Reports Server (NTRS)

Vaughan, Mark A.; Trepte, Charles R.; Winker, David M.; Avery, Melody A.; Campbell, James; Hoff, Ray; Young, Stuart; Getzewich, Brian J.; Tackett, Jason L.; Kar, Jayanta

2011-01-01

The Cloud-Aerosol Lidar and Infrared Pathfinder satellite Observations (CALIPSO) mission was originally conceived and designed as a climate measurements mission, with considerable latency between data acquisition and the release of the level 1 and level 2 data products. However, the unique nature of the CALIPSO lidar backscatter profiles quickly led to the qualitative use of CALIPSO?s near real time (i.e., ? expedited?) lidar data imagery in several different forecasting applications. To enable quantitative use of their near real time analyses, the CALIPSO project recently expanded their expedited data catalog to include all of the standard level 1 and level 2 lidar data products. Also included is a new cloud cleared level 1.5 profile product developed for use by operational forecast centers for verification of aerosol predictions. This paper describes the architecture and content of the CALIPSO expedited data products. The fidelity and accuracy of the expedited products are assessed via comparisons to the standard CALIPSO data products.

Real-time video quality monitoring

NASA Astrophysics Data System (ADS)

Liu, Tao; Narvekar, Niranjan; Wang, Beibei; Ding, Ran; Zou, Dekun; Cash, Glenn; Bhagavathy, Sitaram; Bloom, Jeffrey

2011-12-01

The ITU-T Recommendation G.1070 is a standardized opinion model for video telephony applications that uses video bitrate, frame rate, and packet-loss rate to measure the video quality. However, this model was original designed as an offline quality planning tool. It cannot be directly used for quality monitoring since the above three input parameters are not readily available within a network or at the decoder. And there is a great room for the performance improvement of this quality metric. In this article, we present a real-time video quality monitoring solution based on this Recommendation. We first propose a scheme to efficiently estimate the three parameters from video bitstreams, so that it can be used as a real-time video quality monitoring tool. Furthermore, an enhanced algorithm based on the G.1070 model that provides more accurate quality prediction is proposed. Finally, to use this metric in real-world applications, we present an example emerging application of real-time quality measurement to the management of transmitted videos, especially those delivered to mobile devices.

Intraoperative real-time localization of parathyroid gland with near infrared fluorescence imaging

PubMed Central

Kim, Sung Won; Lee, Hyoung Shin

2017-01-01

Surgeons have cited difficulties in identifying the parathyroid glands (PG) during thyroidectomy. To overcome the limitation of naked eye, many studies on near-infrared fluorescence imaging of PGs have been introduced and suggested that fluorescence imaging is useful for both localizing PGs and evaluating their function. This imaging technique has been reported in two ways: (I) imaging using a fluorescent material called indocyanine green (ICG); and (II) autofluorescence using intrinsic fluorophores. These innovative and novel techniques are expected to have a significant impact on performing thyroid or parathyroid surgery. In this article, current papers that describe ICG fluorescence and autofluorescence imaging of PG during thyroid and parathyroid surgery are reviewed. PMID:29142843

[Measurement of left atrial and ventricular volumes in real-time 3D echocardiography. Validation by nuclear magnetic resonance

NASA Technical Reports Server (NTRS)

Bauer, F.; Shiota, T.; Qin, J. X.; White, R. D.; Thomas, J. D.

2001-01-01

The measurement of the left ventricular ejection fraction is important for the evaluation of cardiomyopathy and depends on the measurement of left ventricular volumes. There are no existing conventional echocardiographic means of measuring the true left atrial and ventricular volumes without mathematical approximations. The aim of this study was to test anew real time 3-dimensional echocardiographic system of calculating left atrial and ventricular volumes in 40 patients after in vitro validation. The volumes of the left atrium and ventricle acquired from real time 3-D echocardiography in the apical view, were calculated in 7 sections parallel to the surface of the probe and compared with atrial (10 patients) and ventricular (30 patients) volumes calculated by nuclear magnetic resonance with the simpson method and with volumes of water in balloons placed in a cistern. Linear regression analysis showed an excellent correlation between the real volume of water in the balloons and volumes given in real time 3-dimensional echocardiography (y = 0.94x + 5.5, r = 0.99, p < 0.001, D = -10 +/- 4.5 ml). A good correlation was observed between real time 3-dimensional echocardiography and nuclear magnetic resonance for the measurement of left atrial and ventricular volumes (y = 0.95x - 10, r = 0.91, p < 0.001, D = -14.8 +/- 19.5 ml and y = 0.87x + 10, r = 0.98, P < 0.001, D = -8.3 +/- 18.7 ml, respectively. The authors conclude that real time three-dimensional echocardiography allows accurate measurement of left heart volumes underlying the clinical potential of this new 3-D method.

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

PubMed Central

Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E.

2016-01-01

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these

Detection of radiation-induced brain necrosis in live rats using label-free time-resolved fluorescence spectroscopy (TRFS) (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hartl, Brad A.; Ma, Htet S. W.; Sridharan, Shamira; Hansen, Katherine; Klich, Melanie; Perks, Julian; Kent, Michael; Kim, Kyoungmi; Fragoso, Ruben; Marcu, Laura

2017-02-01

Differentiating radiation-induced necrosis from recurrent tumor in the brain remains a significant challenge to the neurosurgeon. Clinical imaging modalities are not able to reliably discriminate the two tissue types, making biopsy location selection and surgical management difficult. Label-free fluorescence lifetime techniques have previously been shown to be able to delineate human brain tumor from healthy tissues. Thus, fluorescence lifetime techniques represent a potential means to discriminate the two tissues in real-time during surgery. This study aims to characterize the endogenous fluorescence lifetime signatures from radiation induced brain necrosis in a tumor-free rat model. Fischer rats received a single fraction of 60 Gy of radiation to the right hemisphere using a linear accelerator. Animals underwent a terminal live surgery after gross necrosis had developed, as verified with MRI. During surgery, healthy and necrotic brain tissue was measured with a fiber optic needle connected to a multispectral fluorescence lifetime system. Measurements of the necrotic tissue showed a 48% decrease in intensity and 20% increase in lifetimes relative to healthy tissue. Using a support vector machine classifier and leave-one-out validation technique, the necrotic tissue was correctly classified with 94% sensitivity and 97% specificity. Spectral contribution analysis also confirmed that the primary source of fluorescence contrast lies within the redox and bound-unbound population shifts of nicotinamide adenine dinucleotide. A clinical trial is presently underway to measure these tissue types in humans. These results show for the first time that radiation-induced necrotic tissue in the brain contains significantly different metabolic signatures that are detectable with label-free fluorescence lifetime techniques.

Toward Real-Time Classification of Wake Regimes from Sensor Measurements

NASA Astrophysics Data System (ADS)

Wang, Mengying; Hemati, Maziar S.

2017-11-01

Hydrodynamic signals can transmit information that can be used by marine swimmers to detect disturbances in the local environment. Biological swimmers are able to sense and detect these signals with their hydrodynamic receptor systems. Recently, similar flow sensing systems have been developed with an aim to improve swimming efficiency in human-engineered underwater vehicles. A key part of the sensing strategy is to first classify wake structures in the external fluid, then to execute suitable control actions accordingly. In our previous work, we showed that a variety of 2S and 2P wakes can be distinguished based on time signatures of surface sensor measurements. However, we assumed access to the full dataset. In this talk, we extend our previous findings to classify wake regimes from sensor measurements in real-time, using a recursive Fast Fourier Transform algorithm. Wakes in different dynamical regimes, which may also vary in time, can be distinguished using our approach. Our results provide insights for enhancing hydrodynamic sensory capabilities in human-engineered systems.

A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

PubMed Central

Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible. PMID:24822188

Rapid real-time diagnostic PCR for Trichophyton rubrum and Trichophyton mentagrophytes in patients with tinea unguium and tinea pedis using specific fluorescent probes.

PubMed

Miyajima, Yoshiharu; Satoh, Kazuo; Uchida, Takao; Yamada, Tsuyoshi; Abe, Michiko; Watanabe, Shin-ichi; Makimura, Miho; Makimura, Koichi

2013-03-01

Trichophyton rubrum and Trichophyton mentagrophytes human-type (synonym, Trichophyton interdigitale (anthropophilic)) are major causative pathogens of tinea unguium. For suitable diagnosis and treatment, rapid and accurate identification of etiologic agents in clinical samples using reliable molecular based method is required. For identification of organisms causing tinea unguium, we developed a new real-time polymerase chain reaction (PCR) with a pan-fungal primer set and probe, as well as specific primer sets and probes for T. rubrum and T. mentagrophytes human-type. We designed two sets of primers from the internal transcribed spacer 1 (ITS1) region of fungal ribosomal DNA (rDNA) and three quadruple fluorescent probes, one for detection wide range pathogenic fungi and two for classification of T. rubrum and T. mentagrophytes by specific binding to different sites in the ITS1 region. We investigated the specificity of these primer sets and probes using fungal genomic DNA, and also examined 42 clinical specimens with our real-time PCR. The primers and probes specifically detected T. rubrum, T. mentagrophytes, and a wide range of pathogenic fungi. The causative pathogens were identified in 42 nail and skin samples from 32 patients. The total time required for identification of fungal species in each clinical specimen was about 3h. The copy number of each fungal DNA in the clinical specimens was estimated from the intensity of fluorescence simultaneously. This PCR system is one of the most rapid and sensitive methods available for diagnosing dermatophytosis, including tinea unguium and tinea pedis. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Real-Time CORBA

DTIC Science & Technology

2000-10-01

control systems and prototyped the approach by porting the ILU ORB from Xerox to the Lynx real - time operating system . They then provided a distributed...compliant real - time operating system , a real-time ORB, and an ODMG-compliant real-time ODBMS [12]. The MITRE system is an infrastructure for...the server’s local operating system can handle. For instance, on a node controlled by the VXWorks real - time operating system with 256 local

Real-time photo-magnetic imaging.

PubMed

Nouizi, Farouk; Erkol, Hakan; Luk, Alex; Unlu, Mehmet B; Gulsen, Gultekin

2016-10-01

We previously introduced a new high resolution diffuse optical imaging modality termed, photo-magnetic imaging (PMI). PMI irradiates the object under investigation with near-infrared light and monitors the variations of temperature using magnetic resonance thermometry (MRT). In this paper, we present a real-time PMI image reconstruction algorithm that uses analytic methods to solve the forward problem and assemble the Jacobian matrix much faster. The new algorithm is validated using real MRT measured temperature maps. In fact, it accelerates the reconstruction process by more than 250 times compared to a single iteration of the FEM-based algorithm, which opens the possibility for the real-time PMI.

Real-time interferometric diagnostics of rubidium plasma

NASA Astrophysics Data System (ADS)

Djotyan, G. P.; Bakos, J. S.; Kedves, M. Á.; Ráczkevi, B.; Dzsotjan, D.; Varga-Umbrich, K.; Sörlei, Zs.; Szigeti, J.; Ignácz, P.; Lévai, P.; Czitrovszky, A.; Nagy, A.; Dombi, P.; Rácz, P.

2018-03-01

A method of interferometric real-time diagnostics is developed and applied to rubidium plasma created by strong laser pulses in the femtosecond duration range at different initial rubidium vapor densities using a Michelson-type interferometer. A cosine fit with an exponentially decaying relative phase is applied to the obtained time-dependent interferometry signals to measure the density-length product of the created plasma and its recombination time constant. The presented technique may be applicable for real-time measurements of rubidium plasma dynamics in the AWAKE experiment at CERN, as well as for real-time diagnostics of plasmas created in different gaseous media and on surfaces of solid targets.

Real-time measurement of dust in the workplace using video exposure monitoring: Farming to pharmaceuticals

NASA Astrophysics Data System (ADS)

Walsh, P. T.; Forth, A. R.; Clark, R. D. R.; Dowker, K. P.; Thorpe, A.

2009-02-01

Real-time, photometric, portable dust monitors have been employed for video exposure monitoring (VEM) to measure and highlight dust levels generated by work activities, illustrate dust control techniques, and demonstrate good practice. Two workplaces, presenting different challenges for measurement, were used to illustrate the capabilities of VEM: (a) poultry farming activities and (b) powder transfer operations in a pharmaceutical company. For the poultry farm work, the real-time monitors were calibrated with respect to the respirable and inhalable dust concentrations using cyclone and IOM reference samplers respectively. Different rankings of exposure for typical activities were found on the small farm studied here compared to previous exposure measurements at larger poultry farms: these were mainly attributed to the different scales of operation. Large variations in the ratios of respirable, inhalable and real-time monitor TWA concentrations of poultry farm dust for various activities were found. This has implications for the calibration of light-scattering dust monitors with respect to inhalable dust concentration. In the pharmaceutical application, the effectiveness of a curtain barrier for dust control when dispensing powder in a downflow booth was rapidly demonstrated.

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.

Continuous-flow water sampler for real-time isotopic water measurements

NASA Astrophysics Data System (ADS)

Carter, J.; Dennis, K.

2013-12-01

Measuring the stable isotopes of liquid water (δ18O and δD) is a tool familiar to many Earth scientists, but most current techniques require discrete sampling. For example, isotope ratio mass spectrometry requires the collection of aliquots of water that are then converted to CO2, CO or H2 for analysis. Similarly, laser-based techniques, such as Cavity Ring-Down Spectroscopy (CRDS) convert discrete samples (typically < 2μL) of liquid water to water vapor using a flash vaporization process. By requiring the use of discrete samples fine-scale spatial and temporal studies of changes in δ18O and δD are limited. Here we present a continuous-flow water sampler that will enable scientists to probe isotopic changes in real-time, with applications including, but not limited to, quantification of the 'amount effect' (Dansgaard, 1964) during an individual precipitation event or storm track, real-time mixing of water in river systems, and shipboard continuous water measurements (Munksgaard et al., 2012). Due to the inherent ability of CRDS to measure a continuous flow of water vapor it is an ideal candidate for interfacing with a continuous water sampling system. Here we present results from the first commercially available continuous-flow water sampler, developed by engineers at Picarro. This peripheral device is compatible with Picarro CRDS isotopic water analyzers, allowing real-time, continuous isotopic measurements of liquid water. The new device, which expands upon the design of Munskgaard et al. (2011), utilizes expanded polytetrafluoroethylene (ePTFE) membrane technology to continuously generate gas-phase water, while liquid water is pumped through the system. The water vapor subsequently travels to the CRDS analyzer where the isotopic ratios are measured and recorded. The generation of water vapor using membrane technology is sensitive to environmental conditions, which if not actively control, lead to sustainable experimental noise and drift. Consequently, our

Paramyxovirus fusion: Real-time measurement of parainfluenza virus 5 virus-cell fusion

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

Connolly, Sarah A.; Lamb, Robert A.

2006-11-25

Although cell-cell fusion assays are useful surrogate methods for studying virus fusion, differences between cell-cell and virus-cell fusion exist. To examine paramyxovirus fusion in real time, we labeled viruses with fluorescent lipid probes and monitored virus-cell fusion by fluorimetry. Two parainfluenza virus 5 (PIV5) isolates (W3A and SER) and PIV5 containing mutations within the fusion protein (F) were studied. Fusion was specific and temperature-dependent. Compared to many low pH-dependent viruses, the kinetics of PIV5 fusion was slow, approaching completion within several minutes. As predicted from cell-cell fusion assays, virus containing an F protein with an extended cytoplasmic tail (rSV5 F551)more » had reduced fusion compared to wild-type virus (W3A). In contrast, virus-cell fusion for SER occurred at near wild-type levels, despite the fact that this isolate exhibits a severely reduced cell-cell fusion phenotype. These results support the notion that virus-cell and cell-cell fusion have significant differences.« less

Low-cost, efficient wireless intelligent sensors (LEWIS) measuring real-time reference-free dynamic displacements

NASA Astrophysics Data System (ADS)

Ozdagli, A. I.; Liu, B.; Moreu, F.

2018-07-01

According to railroad managers, displacement of railroad bridges under service loads is an important parameter in the condition assessment and performance evaluation. However, measuring bridge responses in the field is often costly and labor-intensive. This paper proposes a low-cost, efficient wireless intelligent sensor (LEWIS) platform that can compute in real-time the dynamic transverse displacements of railroad bridges under service loads. This sensing platform drives on an open-source Arduino ecosystem and combines low-cost microcontrollers with affordable accelerometers and wireless transmission modules. The proposed LEWIS system is designed to reconstruct dynamic displacements from acceleration measurements onboard, eliminating the need for offline post-processing, and to transmit the data in real-time to a base station where the inspector at the bridge can see the displacements while the train is crossing, or to a remote office if so desired by internet. Researchers validated the effectiveness of the new LEWIS by conducting a series of laboratory experiments. A shake table setup simulated transverse bridge displacements measured on the field and excited the proposed platform, a commercially available wired expensive accelerometer, and reference LVDT displacement sensor. The responses obtained from the wireless system were compared to the displacements reconstructed from commercial accelerometer readings and the reference LVDT. The results of the laboratory experiments demonstrate that the proposed system is capable of reconstructing transverse displacements of railroad bridges under revenue service traffic accurately and transmitting the data in real-time wirelessly. In conclusion, the platform presented in this paper can be used in the performance assessment of railroad bridge network cost-effectively and accurately. Future work includes collecting real-time reference-free displacements of one railroad bridge in Colorado under train crossings to further

Measuring thermodynamic details of DNA hybridization using fluorescence.

PubMed

You, Yong; Tataurov, Andrey V; Owczarzy, Richard

2011-07-01

Modern real-time PCR systems make it easy to monitor fluorescence while temperature is varied for hundreds of samples in parallel, permitting high-throughput studies. We employed such system to investigate melting transitions of ordered nucleic acid structures into disordered random coils. Fluorescent dye and quencher were attached to oligonucleotides in such a way that changes of fluorescence intensity with temperature indicated progression of denaturation. When fluorescence melting data were compared with traditional ultraviolet optical experiments, commonly used dye/quencher combinations, like fluorescein and tetramethylrhodamine, showed substantial discrepancies. We have therefore screened 22 commercially available fluorophores and quenchers for their ability to reliably report annealing and melting transitions. Dependence of fluorescence on temperature and pH was also investigated. The optimal performance was observed using Texas Red or ROX dyes with Iowa Black RQ or Black Hole quenchers. These labels did not alter two-state nature of duplex melting process and provided accurate melting temperatures, free energies, enthalpies, and entropies. We also suggest a new strategy for determination of DNA duplex thermodynamics where concentration of a dye-labeled strand is kept constant and its complementary strand modified with a quencher is added at increasing excess. These methodological improvements will help build predictive models of nucleic acid hybridization. Copyright © 2011 Wiley Periodicals, Inc., a Wiley company.

Toward real-time performance benchmarks for Ada

NASA Technical Reports Server (NTRS)

Clapp, Russell M.; Duchesneau, Louis; Volz, Richard A.; Mudge, Trevor N.; Schultze, Timothy

1986-01-01

The issue of real-time performance measurements for the Ada programming language through the use of benchmarks is addressed. First, the Ada notion of time is examined and a set of basic measurement techniques are developed. Then a set of Ada language features believed to be important for real-time performance are presented and specific measurement methods discussed. In addition, other important time related features which are not explicitly part of the language but are part of the run-time related features which are not explicitly part of the language but are part of the run-time system are also identified and measurement techniques developed. The measurement techniques are applied to the language and run-time system features and the results are presented.

A Portable Array-Type Optical Fiber Sensing Instrument for Real-Time Gas Detection

PubMed Central

Hung, San-Shan; Chang, Hsing-Cheng; Chang, I-Nan

2016-01-01

A novel optical fiber array-type of sensing instrument with temperature compensation for real-time detection was developed to measure oxygen, carbon dioxide, and ammonia simultaneously. The proposed instrument is multi-sensing array integrated with real-time measurement module for portable applications. The sensing optical fibers were etched and polished before coating to increase sensitivities. The ammonia and temperature sensors were each composed of a dye-coated single-mode fiber with constructing a fiber Bragg grating and a long-period filter grating for detecting light intensity. Both carbon dioxide and oxygen sensing structures use multimode fibers where 1-hydroxy-3,6,8-pyrene trisulfonic acid trisodium salt is coated for carbon dioxide sensing and Tris(2,2′-bipyridyl) dichlororuthenium(II) hexahydrate and Tris(bipyridine)ruthenium(II) chloride are coated for oxygen sensing. Gas-induced fluorescent light intensity variation was applied to detect gas concentration. The portable gas sensing array was set up by integrating with photo-electronic measurement modules and a human-machine interface to detect gases in real time. The measured data have been processed using piecewise-linear method. The sensitivity of the oxygen sensor were 1.54%/V and 9.62%/V for concentrations less than 1.5% and for concentrations between 1.5% and 6%, respectively. The sensitivity of the carbon dioxide sensor were 8.33%/V and 9.62%/V for concentrations less than 2% and for concentrations between 2% and 5%, respectively. For the ammonia sensor, the sensitivity was 27.78%/V, while ammonia concentration was less than 2%. PMID:27941636

A Portable Array-Type Optical Fiber Sensing Instrument for Real-Time Gas Detection.

PubMed

Hung, San-Shan; Chang, Hsing-Cheng; Chang, I-Nan

2016-12-08

A novel optical fiber array-type of sensing instrument with temperature compensation for real-time detection was developed to measure oxygen, carbon dioxide, and ammonia simultaneously. The proposed instrument is multi-sensing array integrated with real-time measurement module for portable applications. The sensing optical fibers were etched and polished before coating to increase sensitivities. The ammonia and temperature sensors were each composed of a dye-coated single-mode fiber with constructing a fiber Bragg grating and a long-period filter grating for detecting light intensity. Both carbon dioxide and oxygen sensing structures use multimode fibers where 1-hydroxy-3,6,8-pyrene trisulfonic acid trisodium salt is coated for carbon dioxide sensing and Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate and Tris(bipyridine)ruthenium(II) chloride are coated for oxygen sensing. Gas-induced fluorescent light intensity variation was applied to detect gas concentration. The portable gas sensing array was set up by integrating with photo-electronic measurement modules and a human-machine interface to detect gases in real time. The measured data have been processed using piecewise-linear method. The sensitivity of the oxygen sensor were 1.54%/V and 9.62%/V for concentrations less than 1.5% and for concentrations between 1.5% and 6%, respectively. The sensitivity of the carbon dioxide sensor were 8.33%/V and 9.62%/V for concentrations less than 2% and for concentrations between 2% and 5%, respectively. For the ammonia sensor, the sensitivity was 27.78%/V, while ammonia concentration was less than 2%.

Optimized quantum sensing with a single electron spin using real-time adaptive measurements.

PubMed

Bonato, C; Blok, M S; Dinani, H T; Berry, D W; Markham, M L; Twitchen, D J; Hanson, R

2016-03-01

Quantum sensors based on single solid-state spins promise a unique combination of sensitivity and spatial resolution. The key challenge in sensing is to achieve minimum estimation uncertainty within a given time and with high dynamic range. Adaptive strategies have been proposed to achieve optimal performance, but their implementation in solid-state systems has been hindered by the demanding experimental requirements. Here, we realize adaptive d.c. sensing by combining single-shot readout of an electron spin in diamond with fast feedback. By adapting the spin readout basis in real time based on previous outcomes, we demonstrate a sensitivity in Ramsey interferometry surpassing the standard measurement limit. Furthermore, we find by simulations and experiments that adaptive protocols offer a distinctive advantage over the best known non-adaptive protocols when overhead and limited estimation time are taken into account. Using an optimized adaptive protocol we achieve a magnetic field sensitivity of 6.1 ± 1.7 nT Hz(-1/2) over a wide range of 1.78 mT. These results open up a new class of experiments for solid-state sensors in which real-time knowledge of the measurement history is exploited to obtain optimal performance.

Real-time high dynamic range laser scanning microscopy

PubMed Central

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-01-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging. PMID:27032979

Real time in situ detection of organic nitrates in atmospheric aerosols.

PubMed

Rollins, Andrew W; Smith, Jared D; Wilson, Kevin R; Cohen, Ronald C

2010-07-15

A novel instrument is described that quantifies total particle-phase organic nitrates in real time with a detection limit of 0.11 microg m(-3) min(-1), 45 ppt min(-1) (-ONO(2)). Aerosol nitrates are separated from gas-phase nitrates with a short residence time activated carbon denuder. Detection of organic molecules containing -ONO(2) subunits is accomplished using thermal dissociation coupled to laser induced fluorescence detection of NO(2). This instrument is capable of high time resolution (seconds) measurements of particle-phase organic nitrates, without interference from inorganic nitrate. Here we use it to quantify organic nitrates in secondary organic aerosol generated from high-NO(x) photooxidation of limonene, alpha-pinene, Delta-3-carene, and tridecane. In these experiments the organic nitrate moiety is observed to be 6-15% of the total SOA mass.

Time-synchronized continuous wave laser-induced fluorescence on an oscillatory xenon discharge.

PubMed

MacDonald, N A; Cappelli, M A; Hargus, W A

2012-11-01

A novel approach to time-synchronizing laser-induced fluorescence measurements to an oscillating current in a 60 Hz xenon discharge lamp using a continuous wave laser is presented. A sample-hold circuit is implemented to separate out signals at different phases along a current cycle, and is followed by a lock-in amplifier to pull out the resulting time-synchronized fluorescence trace from the large background signal. The time evolution of lower state population is derived from the changes in intensity of the fluorescence excitation line shape resulting from laser-induced fluorescence measurements of the 6s(')[1/2](1)(0)-6p(')[3/2](2) xenon atomic transition at λ = 834.68 nm. Results show that the lower state population oscillates at twice the frequency of the discharge current, 120 Hz.

Identification of real-time diagnostic measures of visual distraction with an automatic eye-tracking system.

PubMed

Zhang, Harry; Smith, Matthew R H; Witt, Gerald J

2006-01-01

This study was conducted to identify eye glance measures that are diagnostic of visual distraction. Visual distraction degrades performance, but real-time diagnostic measures have not been identified. In a driving simulator, 14 participants responded to a lead vehicle braking at -2 or -2.7 m/s2 periodically while reading a varying number of words (6-15 words every 13 s) on peripheral displays (with diagonal eccentricities of 24 degrees, 43 degrees, and 75 degrees). As the number of words and display eccentricity increased, total glance duration and reaction time increased and driving performance suffered. Correlation coefficients between several glance measures and reaction time or performance variables were reliably high, indicating that these glance measures are diagnostic of visual distraction. It is predicted that for every 25% increase in total glance duration, reaction time is increased by 0.39 s and standard deviation of lane position is increased by 0.06 m. Potential applications of this research include assessing visual distraction in real time, delivering advisories to distracted drivers to reorient their attention to driving, and using distraction information to adapt forward collision and lane departure warning systems to enhance system effectiveness.

Obtaining Reliable Predictions of Terrestrial Energy Coupling From Real-Time Solar Wind Measurements

NASA Technical Reports Server (NTRS)

Weimer, Daniel R.

2002-01-01

Measurements of the interplanetary magnetic field (IMF) from the ACE (Advanced Composition Explorer), Wind, IMP-8 (Interplanetary Monitoring Platform), and Geotail spacecraft have revealed that the IMF variations are contained in phase planes that are tilted with respect to the propagation direction, resulting in continuously variable changes in propagation times between spacecraft, and therefore, to the Earth. Techniques for using 'minimum variance analysis' have been developed in order to be able to measure the phase front tilt angles, and better predict the actual propagation times from the L1 orbit to the Earth, using only the real-time IMF measurements from one spacecraft. The use of empirical models with the IMF measurements at L1 from ACE (or future satellites) for predicting 'space weather' effects has also been demonstrated.

Real-Time Measurements of Aft Dome Insulation Erosion on Space Shuttle Reusable Solid Rocket Motor

NASA Technical Reports Server (NTRS)

McWhorter, Bruce; Ewing, Mark; Albrechtsen, Kevin; Noble, Todd; Longaker, Matt

2004-01-01

Real-time erosion of aft dome internal insulation was measured with internal instrumentation on a static test of a lengthened version of the Space Shuffle Reusable Solid Rocket Motor (RSRM). This effort marks the first time that real-time aft dome insulation erosion (Le., erosion due to the combined effects of thermochemical ablation and mechanical abrasion) was measured in this kind of large motor static test [designated as Engineering Test Motor number 3 (ETM3)I. This paper presents data plots of the erosion depth versus time. The data indicates general erosion versus time behavior that is in contrast to what would be expected from earlier analyses. Engineers have long known that the thermal environment in the aft dome is severe and that the resulting aft dome insulation erosion is significant. Models of aft dome erosion involve a two-step process of computational fluid dynamics (CFD) modeling and material ablation modeling. This modeling effort is complex. The time- dependent effects are difficult to verify with only prefire and postfire insulation measurements. Nozzle vectoring, slag accumulation, and changing boundary conditions will affect the time dependence of aft dome erosion. Further study of this data and continued measurements on future motors will increase our understanding of the aft dome flow and erosion environment.

Combination of optical shape measurement and augmented reality for task support: II. Real-time feedback of shape measurement results

NASA Astrophysics Data System (ADS)

Yamauchi, Makoto; Iwamoto, Kazuyo

2010-05-01

Line heating is a skilled task in shipbuilding to shape the outer plates of ship hulls. Real-time information on the deformation of the plates during the task would be helpful to workers performing this process. Therefore, we herein propose an interactive scheme for supporting workers performing line heating; the system provides such information through an optical shape measurement instrument combined with an augmented reality (AR) system. The instrument was designed and fabricated so that the measured data were represented using coordinates based on fiducial markers. Since the markers were simultaneously used in the AR system for the purpose of positioning, the data could then be displayed to the workers through a head-mounted display as a virtual image overlaid on the plates. Feedback of the shape measurement results was thus performed in real time using the proposed system.

Real Time Revisited

NASA Astrophysics Data System (ADS)

Allen, Phillip G.

1985-12-01

The call for abolishing photo reconnaissance in favor of real time is once more being heard. Ten years ago the same cries were being heard with the introduction of the Charge Coupled Device (CCD). The real time system problems that existed then and stopped real time proliferation have not been solved. The lack of an organized program by either DoD or industry has hampered any efforts to solve the problems, and as such, very little has happened in real time in the last ten years. Real time is not a replacement for photo, just as photo is not a replacement for infra-red or radar. Operational real time sensors can be designed only after their role has been defined and improvements made to the weak links in the system. Plodding ahead on a real time reconnaissance suite without benefit of evaluation of utility will allow this same paper to be used ten years from now.

Glowing clay: Real time tracing using a suite of novel clay based fluorescent tracers

NASA Astrophysics Data System (ADS)

Hardy, Robert; Quinton, John; Pates, Jackie; Coogan, Mike

2015-04-01

Clay is one of the most mobile fractions of soil due to its small particle size. It is also known to sorb many chemicals, such as nutrients (notably phosphorus), agrochemicals and heavy metals. The movement of clay is therefore linked with the transport and fate of these substances. A novel fluorescent clay tracing suite has been produced, together with an imaging technique. This suite consists of qualitative clay tracers, using rhodamine based fluorophores, and quantitative clay tracers, using metal based fluorophores. Efforts have also been made to allow integration of commercially available tracers, which are silt and sand sized. The clay tracers exploit the high affinity that montmorillonite has for Rhodamine B and Ru(bpy)3. This allows for an extremely thin layer of the fluorophore to be sorbed onto the clay's surface, in much that same way as materials in the natural environment will bind to clay. The tracer that is produced retains key chemical and physical properties of clay, such as size, shape and density. The retention of these micro-properties results in the retention of macro-properties, such as tendency to aggregate and cracking on drying. Imaging techniques have been developed to analyse these tracers. The imaging system uses diffused laser light to excite the tracer and a modified DSLR camera to image the soil surface. The images have been compiled into a time lapse video showing the movement of clay over the course of a rainfall event. This is the first time that the quantitative movement of clay has been recorded over a soil surface in real time. 4D data can be extracted from the images allowing the spatial location and intensity of tracer to be monitored over time, with mm precision and on the timescale of seconds. As the system can also work with a commercial tracer it is possible to investigate the movement of particles of almost any size and over a range of scales from soil box to hillside. This allows users to access this technique without

Interaction of on-site and near real time measured turbidity and enzyme activity in stream water.

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Farnleitner, Andreas H.; Zessner, Matthias

2013-04-01

On-site and on-line systems that provide an integrated surveillance of physicochemical and microbiological parameters gain significance in water quality monitoring. Particular relating to diffuse pollution from agricultural areas and use-orientated protection of waters the detection of faecal pollution is a fundamental part. For the near real time and on-site detection of microbiological faecal pollution of water, the beta-D- Glucuronidase (GLUC) enzymatic activity has been suggested as a surrogate parameter. Due to possible short measure intervals of three hours, this method has high potential as a water quality monitoring tool. While cultivation based standard determination takes more than one working day (Cabral 2010) the potential advantage of detecting the GLUC activity is the high temporal measuring resolution. Yet, there is still a big gap of knowledge on the sensitivity and specificity concerning the faecal indication capacity of GLUC in relation to standard assays (Cabral 2010). Interference effects of physicochemical parameters on the enzymatic activity respectively fluorescence have been discussed (Molina-Munoz et al. 2007; Tryland and Fiksdal 1998, Biswal et al. 2003). Results from a monitoring of a rivulet in an agricultural catchment in Lower Austria (HOAL - Hydrological Open Air Laboratory) are presented here. The HOAL offers technical resources that allow measurements at high temporal and spatial resolution and to apply various hydrological methods in one catchment. Two automated enzymatic measuring devices (Coliguard, mbOnline, Austria) and physicochemical in-stream measurements are used, as well as in-stream spectroscopy (spectrolyser, s::can, Austria). Accuracy of both enzymatic measuring devices is compared through diverse hydrological and seasonal conditions. Reference analyses by cultivation based determination were performed. Data from Coliguard devices is combined with physicochemical and spectroscopy data to gain information about the

Towards real time spatially resolved data on sediment transport: 1) tracing the motion of the fluorescent soil particles under rainfall

NASA Astrophysics Data System (ADS)

Quinton, John; Hardy, Rob; Pates, Jackie; James, Mike

2017-04-01

Understanding where sediment originates from and where it travels to, in what quantities and at which rate is at the heart of many questions surrounding sediment transport, including the connectivity problem. Progress towards unravelling these questions and deepening our understanding has come from a wide range of approaches, including laboratory and field experiments conducted at a variety of scales. In seeking to understand the connectivity of sources and sinks of sediment scientists have spent considerable energy in developing tracing technologies. These have included numerous studies that have relied on the chemical properties of the soil and sediment to establish source-sink connectivity, and the use of 137Ceasium, from radioactive fall-out, to map sediment redistribution. More recently there has been an upsurge in interest in the use of artificially applied soil tracers, including rare earth element oxides and magnetic minerals. However all these tracing methods have a significant drawback: they rely on the collection of samples to assess their concentration. This means that their spatial distribution cannot easily be established in situ and that the environment that is being studied is damaged by the sampling process; nor can data be collected in real time which allows a dynamic understanding of erosion and transport processes to be developed. In this paper we present a methodology for use with a commercially available fluorescent tracer. The tracer is produced in a range of sizes and fluorescent signatures and can be applied to the soil surface. Here we report on an application that combines novel fluorescent videography techniques with custom image processing to trace the motion of the fluorescent soil particles under rainfall. Here we demonstrate the tracking of multiple sub-millimetre particles simultaneously, establishing their position 50 times a second with submillimetre precision. From this we are able to visualise and quantify parameters such as

Detecting aromatic compounds on planetary surfaces using ultraviolet time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Eshelman, E.; Daly, M. G.; Slater, G.; Cloutis, E.

2018-02-01

Many aromatic organic molecules exhibit strong and characteristic fluorescence when excited with ultraviolet radiation. As laser excitation in the ultraviolet generates both fluorescence and resonantly enhanced Raman scattering of aromatic vibrational modes, combined Raman and fluorescence instruments have been proposed to search for organic compounds on Mars. In this work the time-resolved fluorescence of a suite of 24 compounds composed of 2-5 ringed alternant, non-alternant, and heterocyclic PAHs was measured. Fluorescence instrumentation with similar specifications to a putative flight instrument was capable of observing the fluorescence decay of these compounds with a sub-ns resolution. Incorporating time-resolved capabilities was also found to increase the ability to discriminate between individual PAHs. Incorporating time-resolved fluorescence capabilities into an ultraviolet gated Raman system intended for a rover or lander can increase the ability to detect and characterize PAHs on planetary surfaces.

Optimized Time-Gated Fluorescence Spectroscopy for the Classification and Recycling of Fluorescently Labeled Plastics.

PubMed

Fomin, Petr; Zhelondz, Dmitry; Kargel, Christian

2017-05-01

For the production of high-quality parts from recycled plastics, a very high purity of the plastic waste to be recycled is mandatory. The incorporation of fluorescent tracers ("markers") into plastics during the manufacturing process helps overcome typical problems of non-tracer based optical classification methods. Despite the unique emission spectra of fluorescent markers, the classification becomes difficult when the host plastics exhibit (strong) autofluorescence that spectrally overlaps the marker fluorescence. Increasing the marker concentration is not an option from an economic perspective and might also adversely affect the properties of the plastics. A measurement approach that suppresses the autofluorescence in the acquired signal is time-gated fluorescence spectroscopy (TGFS). Unfortunately, TGFS is associated with a lower signal-to-noise (S/N) ratio, which results in larger classification errors. In order to optimize the S/N ratio we investigate and validate the best TGFS parameters-derived from a model for the fluorescence signal-for plastics labeled with four specifically designed fluorescent markers. In this study we also demonstrate the implementation of TGFS on a measurement and classification prototype system and determine its performance. Mean values for a sensitivity of [Formula: see text] = 99.93% and precision [Formula: see text] = 99.80% were achieved, proving that a highly reliable classification of plastics can be achieved in practice.

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.

Diagnosis of meningioma by time-resolved fluorescence spectroscopy.

PubMed

Butte, Pramod V; Pikul, Brian K; Hever, Aviv; Yong, William H; Black, Keith L; Marcu, Laura

2005-01-01

We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors.

Diagnosis of meningioma by time-resolved fluorescence spectroscopy

PubMed Central

Butte, Pramod V.; Pikul, Brian K.; Hever, Aviv; Yong, William H.; Black, Keith L.; Marcu, Laura

2010-01-01

We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors. PMID:16409091

Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays.

PubMed

Durtschi, Jacob D; Stevenson, Jeffery; Hymas, Weston; Voelkerding, Karl V

2007-02-01

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.

Development of a Capacitive Ice Sensor to Measure Ice Growth in Real Time

PubMed Central

Zhi, Xiang; Cho, Hyo Chang; Wang, Bo; Ahn, Cheol Hee; Moon, Hyeong Soon; Go, Jeung Sang

2015-01-01

This paper presents the development of the capacitive sensor to measure the growth of ice on a fuel pipe surface in real time. The ice sensor consists of pairs of electrodes to detect the change in capacitance and a thermocouple temperature sensor to examine the ice formation situation. In addition, an environmental chamber was specially designed to control the humidity and temperature to simulate the ice formation conditions. From the humidity, a water film is formed on the ice sensor, which results in an increase in capacitance. Ice nucleation occurs, followed by the rapid formation of frost ice that decreases the capacitance suddenly. The capacitance is saturated. The developed ice sensor explains the ice growth providing information about the icing temperature in real time. PMID:25808770

Development of a capacitive ice sensor to measure ice growth in real time.

PubMed

Zhi, Xiang; Cho, Hyo Chang; Wang, Bo; Ahn, Cheol Hee; Moon, Hyeong Soon; Go, Jeung Sang

2015-03-19

This paper presents the development of the capacitive sensor to measure the growth of ice on a fuel pipe surface in real time. The ice sensor consists of pairs of electrodes to detect the change in capacitance and a thermocouple temperature sensor to examine the ice formation situation. In addition, an environmental chamber was specially designed to control the humidity and temperature to simulate the ice formation conditions. From the humidity, a water film is formed on the ice sensor, which results in an increase in capacitance. Ice nucleation occurs, followed by the rapid formation of frost ice that decreases the capacitance suddenly. The capacitance is saturated. The developed ice sensor explains the ice growth providing information about the icing temperature in real time.

IoT for Real-Time Measurement of High-Throughput Liquid Dispensing in Laboratory Environments.

PubMed

Shumate, Justin; Baillargeon, Pierre; Spicer, Timothy P; Scampavia, Louis

2018-04-01

Critical to maintaining quality control in high-throughput screening is the need for constant monitoring of liquid-dispensing fidelity. Traditional methods involve operator intervention with gravimetric analysis to monitor the gross accuracy of full plate dispenses, visual verification of contents, or dedicated weigh stations on screening platforms that introduce potential bottlenecks and increase the plate-processing cycle time. We present a unique solution using open-source hardware, software, and 3D printing to automate dispenser accuracy determination by providing real-time dispense weight measurements via a network-connected precision balance. This system uses an Arduino microcontroller to connect a precision balance to a local network. By integrating the precision balance as an Internet of Things (IoT) device, it gains the ability to provide real-time gravimetric summaries of dispensing, generate timely alerts when problems are detected, and capture historical dispensing data for future analysis. All collected data can then be accessed via a web interface for reviewing alerts and dispensing information in real time or remotely for timely intervention of dispense errors. The development of this system also leveraged 3D printing to rapidly prototype sensor brackets, mounting solutions, and component enclosures.

Can activity within the external abdominal oblique be measured using real-time ultrasound imaging?

PubMed

John, E K; Beith, I D

2007-11-01

Differences in the function of the anterolateral abdominal muscles have been the subject of much investigation, but primarily using electromyography. Recently changes in thickness of transversus abdominis and internal oblique measured from real-time ultrasound images have been shown to represent activity within these muscles. However it is still unclear if such a change in thickness in external oblique similarly represents activity within that muscle. The purpose of this study was to investigate the relationship between change in thickness and muscle activity in the external oblique using real-time ultrasound and surface electromyography. Simultaneous measurements of electromyography and real-time ultrasound images of external oblique were studied in up to 24 subjects during two tasks compared to the muscle at rest (1) isometric trunk rotation and (2) drawing in the lower abdomen. Changes in muscle thickness correlated significantly with electromyography during isometric trunk rotation in the majority of subjects but with a significant difference between subjects. In contrast, the relationship between change in thickness and electrical activity in the muscle when drawing in the lower abdomen was significant in less than 50% of subjects and the muscle often got thinner. Thickness changes of external oblique can be used as a valid indicator of electromyography activity during isometric trunk rotation, though the relationship is not as good as previously published data for transversus abdominis. Thickness changes of external oblique measured during lower abdominal drawing in cannot be used to detect activity within this muscle.

System and method for measuring fluorescence of a sample

DOEpatents

Riot, Vincent J

2015-03-24

The present disclosure provides a system and a method for measuring fluorescence of a sample. The sample may be a polymerase-chain-reaction (PCR) array, a loop-mediated-isothermal amplification array, etc. LEDs are used to excite the sample, and a photodiode is used to collect the sample's fluorescence. An electronic offset signal is used to reduce the effects of background fluorescence and the noises from the measurement system. An integrator integrates the difference between the output of the photodiode and the electronic offset signal over a given period of time. The resulting integral is then converted into digital domain for further processing and storage.

Brain physiological state evaluated by real-time multiparametric tissue spectroscopy in vivo

NASA Astrophysics Data System (ADS)

Mayevsky, Avraham; Barbiro-Michaely, Efrat; Kutai-Asis, Hofit; Deutsch, Assaf; Jaronkin, Alex

2004-07-01

The significance of normal mitochondrial function in cellular energy homeostasis as well as its involvement in acute and chronic neurodegenerative disease was reviewed recently (Nicholls & Budd. Physiol Rev. 80: 315-360, 2000). Nevertheless, monitoring of mitochondrial function in vivo and real time mode was not used by many investigators and is very rare in clinical practice. The main principle tool available for the evaluation of mitochondrial function is the monitoring of NADH fluorescence. In order to interpret correctly the changes in NADH redox state in vivo, it is necessary to correlate this signal to other parameters, reflecting O2 supply to the brain. Therefore, we have developed and applied a multiparametric optical monitoring system, by which microcirculatory blood flow and hemoglobin oxygenation is measured, together with mitochondrial NADH fluorescence. Since the calibration of these signals is not in absolute units, the simultaneous monitoring provide a practical tool for the interpretation of brain functional state under various pathophysiological conditions. The monitoring system combines a time-sharing fluorometer-reflectometer for the measurement of NADH fluorescence and hemoglobin oxygenation as well as a laser Doppler flowmeter for the recording of microcirculatory blood flow. A combined fiber optic probe was located on the surface of the brain using a skull cemented cannula. Rats and gerbils were exposed to anoxia, ischemia and spreading depression and the functional state of the brain was evaluated. The results showed a clear correlation between O2 supply/demand as well as, energy balance under the various pathophysiological conditions. This monitoring approach could be adapted to clinical monitoring of tissue vitality.

Dynamic tissue analysis using time- and wavelength-resolved fluorescence spectroscopy for atherosclerosis diagnosis

PubMed Central

Sun, Yinghua; Sun, Yang; Stephens, Douglas; Xie, Hongtao; Phipps, Jennifer; Saroufeem, Ramez; Southard, Jeffrey; Elson, Daniel S.; Marcu, Laura

2011-01-01

Simultaneous time- and wavelength-resolved fluorescence spectroscopy (STWRFS) was developed and tested for the dynamic characterization of atherosclerotic tissue ex vivo and arterial vessels in vivo. Autofluorescence, induced by a 337 nm, 700 ps pulsed laser, was split to three wavelength sub-bands using dichroic filters, with each sub-band coupled into a different length of optical fiber for temporal separation. STWRFS allows for fast recording/analysis (few microseconds) of time-resolved fluorescence emission in these sub-bands and rapid scanning. Distinct compositions of excised human atherosclerotic aorta were clearly discriminated over scanning lengths of several centimeters based on fluorescence lifetime and the intensity ratio between 390 and 452 nm. Operation of STWRFS blood flow was further validated in pig femoral arteries in vivo using a single-fiber probe integrated with an ultrasound imaging catheter. Current results demonstrate the potential of STWRFS as a tool for real-time optical characterization of arterial tissue composition and for atherosclerosis research and diagnosis. PMID:21369214

Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.

PubMed

Martinez-Serra, Jordi; Robles, Juan; Nicolàs, Antoni; Gutierrez, Antonio; Ros, Teresa; Amat, Juan Carlos; Alemany, Regina; Vögler, Oliver; Abelló, Aina; Noguera, Aina; Besalduch, Joan

2014-01-01

Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC). In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler(®) 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE). After testing 34 samples comparing the real-time crossing point (CP) values between WBC (5×10(6) WBC/mL) and purified DNA (20 ng/μL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×10(6) WBC/mL) instead of DNA (20 ng/μL), we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification/extraction.

A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy

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

Gong, S.; Labanca, I.; Rech, I.

2014-10-15

Fluorescence correlation spectroscopy (FCS) is a well-established technique to study binding interactions or the diffusion of fluorescently labeled biomolecules in vitro and in vivo. Fast FCS experiments require parallel data acquisition and analysis which can be achieved by exploiting a multi-channel Single Photon Avalanche Diode (SPAD) array and a corresponding multi-input correlator. This paper reports a 32-channel FPGA based correlator able to perform 32 auto/cross-correlations simultaneously over a lag-time ranging from 10 ns up to 150 ms. The correlator is included in a 32 × 1 SPAD array module, providing a compact and flexible instrument for high throughput FCS experiments.more » However, some inherent features of SPAD arrays, namely afterpulsing and optical crosstalk effects, may introduce distortions in the measurement of auto- and cross-correlation functions. We investigated these limitations to assess their impact on the module and evaluate possible workarounds.« less

Optimized quantum sensing with a single electron spin using real-time adaptive measurements

NASA Astrophysics Data System (ADS)

Bonato, C.; Blok, M. S.; Dinani, H. T.; Berry, D. W.; Markham, M. L.; Twitchen, D. J.; Hanson, R.

2016-03-01

Quantum sensors based on single solid-state spins promise a unique combination of sensitivity and spatial resolution. The key challenge in sensing is to achieve minimum estimation uncertainty within a given time and with high dynamic range. Adaptive strategies have been proposed to achieve optimal performance, but their implementation in solid-state systems has been hindered by the demanding experimental requirements. Here, we realize adaptive d.c. sensing by combining single-shot readout of an electron spin in diamond with fast feedback. By adapting the spin readout basis in real time based on previous outcomes, we demonstrate a sensitivity in Ramsey interferometry surpassing the standard measurement limit. Furthermore, we find by simulations and experiments that adaptive protocols offer a distinctive advantage over the best known non-adaptive protocols when overhead and limited estimation time are taken into account. Using an optimized adaptive protocol we achieve a magnetic field sensitivity of 6.1 ± 1.7 nT Hz-1/2 over a wide range of 1.78 mT. These results open up a new class of experiments for solid-state sensors in which real-time knowledge of the measurement history is exploited to obtain optimal performance.

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.

MIMO-OFDM for a Cellular Deployment - Concepts, Real-Time Implementation and Measurements Towards 3GPP-LTE

DTIC Science & Technology

2007-09-01

CONCEPTS, REAL-TIME IMPLEMENTATION AND MEASUREMENTS TOWARDS 3GPP-LTE T. Haustein , J. Eichinger, W. Zirwas, E. Schulz Nokia Siemens...BER (bottom) in an office scenario while the UE is moved from one room to another. REFERENCES [1] V. Jungnickel, A. Forck, T. Haustein , C. Juchems...2.12.2006 [3] T. Haustein , A. Forck, H. Gäbler, V. Jungnickel and S. Schif- fermüller, „Real-Time Experiments on Channel Adaptive Transmis- sion in

A handheld real time thermal cycler for bacterial pathogen detection.

PubMed

Higgins, James A; Nasarabadi, Shanavaz; Karns, Jeffrey S; Shelton, Daniel R; Cooper, Mary; Gbakima, Aiah; Koopman, Ronald P

2003-08-15

The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002.

Magnetostrictive patch sensor system for battery-less real-time measurement of torsional vibrations of rotating shafts

NASA Astrophysics Data System (ADS)

Lee, Jun Kyu; Seung, Hong Min; Park, Chung Il; Lee, Joo Kyung; Lim, Do Hyeong; Kim, Yoon Young

2018-02-01

Real-time uninterrupted measurement for torsional vibrations of rotating shafts is crucial for permanent health monitoring. So far, strain gauge systems with telemetry units have been used for real-time monitoring. However, they have a critical disadvantage in that shaft operations must be stopped intermittently to replace telemetry unit batteries. To find an alternative method to carry out battery-less real-time measurement for torsional vibrations of rotating shafts, a magnetostrictive patch sensor system was proposed in the present study. Since the proposed sensor does not use any powered telemetry system, no battery is needed and thus there is no need to stop rotating shafts for battery replacement. The proposed sensor consists of magnetostrictive patches and small magnets tightly bonded onto a shaft. A solenoid coil is placed around the shaft to convert magnetostrictive patch deformation by shaft torsional vibration into electric voltage output. For sensor design and characterization, investigations were performed in a laboratory on relatively small-sized stationary solid shaft. A magnetostrictive patch sensor system was then designed and installed on a large rotating propulsion shaft of an LPG carrier ship in operation. Vibration signals were measured using the proposed sensor system and compared to those measured with a telemetry unit-equipped strain gauge system.

"Fast" Is Not "Real-Time": Designing Effective Real-Time AI Systems

NASA Astrophysics Data System (ADS)

O'Reilly, Cindy A.; Cromarty, Andrew S.

1985-04-01

Realistic practical problem domains (such as robotics, process control, and certain kinds of signal processing) stand to benefit greatly from the application of artificial intelligence techniques. These problem domains are of special interest because they are typified by complex dynamic environments in which the ability to select and initiate a proper response to environmental events in real time is a strict prerequisite to effective environmental interaction. Artificial intelligence systems developed to date have been sheltered from this real-time requirement, however, largely by virtue of their use of simplified problem domains or problem representations. The plethora of colloquial and (in general) mutually inconsistent interpretations of the term "real-time" employed by workers in each of these domains further exacerbates the difficul-ties in effectively applying state-of-the-art problem solving tech-niques to time-critical problems. Indeed, the intellectual waters are by now sufficiently muddied that the pursuit of a rigorous treatment of intelligent real-time performance mandates the redevelopment of proper problem perspective on what "real-time" means, starting from first principles. We present a simple but nonetheless formal definition of real-time performance. We then undertake an analysis of both conventional techniques and AI technology with respect to their ability to meet substantive real-time performance criteria. This analysis provides a basis for specification of problem-independent design requirements for systems that would claim real-time performance. Finally, we discuss the application of these design principles to a pragmatic problem in real-time signal understanding.

Using Biometric Measurement in Real-Time as a Sympathetic System in Computer Games

ERIC Educational Resources Information Center

Charij, Stephanie; Oikonomou, Andreas

2013-01-01

With the increasing potential for gaming hardware and peripherals to support biometrics, their application within the games industry for software and design should be considered. This paper assesses the ability to use a form of biometric measurement, heart rate, in real-time to improve the challenge and enjoyment of a game by catering it to…

High-speed real-time 3-D coordinates measurement based on fringe projection profilometry considering camera lens distortion

NASA Astrophysics Data System (ADS)

Feng, Shijie; Chen, Qian; Zuo, Chao; Sun, Jiasong; Yu, Shi Ling

2014-10-01

Optical three-dimensional (3-D) profilometry is gaining increasing attention for its simplicity, flexibility, high accuracy, and non-contact nature. Recent advances in imaging sensors and digital projection technology further its progress in high-speed, real-time applications, enabling 3-D shapes reconstruction of moving objects and dynamic scenes. However, the camera lens is never perfect and the lens distortion does influence the accuracy of the measurement result, which is often overlooked in the existing real-time 3-D shape measurement systems. To this end, here we present a novel high-speed real-time 3-D coordinates measuring technique based on fringe projection with the consideration of the camera lens distortion. A pixel mapping relation between a distorted image and a corrected one is pre-determined and stored in computer memory for real-time fringe correction. The out-of-plane height is obtained firstly and the acquisition for the two corresponding in-plane coordinates follows on the basis of the solved height. Besides, a method of lookup table (LUT) is introduced as well for fast data processing. Our experimental results reveal that the measurement error of the in-plane coordinates has been reduced by one order of magnitude and the accuracy of the out-plane coordinate been tripled after the distortions being eliminated. Moreover, owing to the generated LUTs, a 3-D reconstruction speed of 92.34 frames per second can be achieved.

Fluorescence imaging of angiogenesis in green fluorescent protein-expressing tumors

NASA Astrophysics Data System (ADS)

Yang, Meng; Baranov, Eugene; Jiang, Ping; Li, Xiao-Ming; Wang, Jin W.; Li, Lingna; Yagi, Shigeo; Moossa, A. R.; Hoffman, Robert M.

2002-05-01

The development of therapeutics for the control of tumor angiogenesis requires a simple, reliable in vivo assay for tumor-induced vascularization. For this purpose, we have adapted the orthotopic implantation model of angiogenesis by using human and rodent tumors genetically tagged with Aequorea victoria green fluorescent protein (GFP) for grafting into nude mice. Genetically-fluorescent tumors can be readily imaged in vivo. The non-luminous induced capillaries are clearly visible against the bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. Fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. High-level GFP-expressing tumor cell lines made it possible to acquire the high-resolution real-time fluorescent optical images of angiogenesis in both primary tumors and their metastatic lesions in various human and rodent tumor models by means of a light-based imaging system. Intravital images of angiogenesis onset and development were acquired and quantified from a GFP- expressing orthotopically-growing human prostate tumor over a 19-day period. Whole-body optical imaging visualized vessel density increasing linearly over a 20-week period in orthotopically-growing, GFP-expressing human breast tumor MDA-MB-435. Vessels in an orthotopically-growing GFP- expressing Lewis lung carcinoma tumor were visualized through the chest wall via a reversible skin flap. These clinically-relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological micro- environments.

Application of real-time digitization techniques in beam measurement for accelerators

NASA Astrophysics Data System (ADS)

Zhao, Lei; Zhan, Lin-Song; Gao, Xing-Shun; Liu, Shu-Bin; An, Qi

2016-04-01

Beam measurement is very important for accelerators. In this paper, modern digital beam measurement techniques based on IQ (In-phase & Quadrature-phase) analysis are discussed. Based on this method and high-speed high-resolution analog-to-digital conversion, we have completed three beam measurement electronics systems designed for the China Spallation Neutron Source (CSNS), Shanghai Synchrotron Radiation Facility (SSRF), and Accelerator Driven Sub-critical system (ADS). Core techniques of hardware design and real-time system calibration are discussed, and performance test results of these three instruments are also presented. Supported by National Natural Science Foundation of China (11205153, 10875119), Knowledge Innovation Program of the Chinese Academy of Sciences (KJCX2-YW-N27), and the Fundamental Research Funds for the Central Universities (WK2030040029),and the CAS Center for Excellence in Particle Physics (CCEPP).

MISR Level 1 Near Real Time Products

Atmospheric Science Data Center

2016-10-31

Level 1 Near Real Time The MISR Near Real Time Level 1 data products ... km MISR swath and projected onto a Space-Oblique Mercator (SOM) map grid. The Ellipsoid-projected and Terrain-projected top-of-atmosphere (TOA) radiance products provide measurements respectively resampled onto the ...

Practical performance of real-time shot-noise measurement in continuous-variable quantum key distribution

NASA Astrophysics Data System (ADS)

Wang, Tao; Huang, Peng; Zhou, Yingming; Liu, Weiqi; Zeng, Guihua

2018-01-01

In a practical continuous-variable quantum key distribution (CVQKD) system, real-time shot-noise measurement (RTSNM) is an essential procedure for preventing the eavesdropper exploiting the practical security loopholes. However, the performance of this procedure itself is not analyzed under the real-world condition. Therefore, we indicate the RTSNM practical performance and investigate its effects on the CVQKD system. In particular, due to the finite-size effect, the shot-noise measurement at the receiver's side may decrease the precision of parameter estimation and consequently result in a tight security bound. To mitigate that, we optimize the block size for RTSNM under the ensemble size limitation to maximize the secure key rate. Moreover, the effect of finite dynamics of amplitude modulator in this scheme is studied and its mitigation method is also proposed. Our work indicates the practical performance of RTSNM and provides the real secret key rate under it.

Near-Infrared II Fluorescence for Imaging Hindlimb Vessel Regeneration with Dynamic Tissue Perfusion Measurement

PubMed Central

Hong, Guosong; Lee, Jerry C.; Jha, Arshi; Diao, Shuo; Nakayama, Karina H.; Hou, Luqia; Doyle, Timothy C.; Robinson, Joshua T.; Antaris, Alexander L.; Dai, Hongjie; Cooke, John P.; Huang, Ngan F.

2014-01-01

Background Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000–1400 nm) of photon wavelengths. Methods and Results Owing to the reduced photon scattering of NIR-II fluorescence compared to traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microCT. Furthermore, imaging over 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P < 0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy. Conclusions The penetration depth of millimeters, high spatial resolution and fast acquisition rate of NIR-II imaging makes it a useful imaging tool for murine models of vascular disease. PMID:24657826

Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement.

PubMed

Hong, Guosong; Lee, Jerry C; Jha, Arshi; Diao, Shuo; Nakayama, Karina H; Hou, Luqia; Doyle, Timothy C; Robinson, Joshua T; Antaris, Alexander L; Dai, Hongjie; Cooke, John P; Huang, Ngan F

2014-05-01

Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000-1400 nm) of photon wavelengths. Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P<0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy. The penetration depth of millimeters, high spatial resolution, and fast acquisition rate of NIR-II imaging make it a useful imaging tool for murine models of vascular disease. © 2014 American Heart Association, Inc.

A fluorescence spectroscopy assay for real-time monitoring of enzyme immobilization into mesoporous silica particles.

PubMed

Nabavi Zadeh, Pegah S; Mallak, Kassam Abdel; Carlsson, Nils; Åkerman, Björn

2015-05-01

Mesoporous silica particles are used as support material for immobilization of enzymes. Here we investigated a fluorescence-based assay for real-time monitoring of the immobilization of lipase, bovine serum albumin, and glucose oxidase into micrometer-sized mesoporous silica particles. The proteins are labeled with the dye epicocconone, and the interaction with the particles is observed as an increase in emission intensity of the protein-dye conjugates that can be quantified if correcting for a comparatively slow photobleaching. The immobilization occurs in tens of minutes to hours depending on particle concentration and type of protein. In the limit of excess particles over proteins, the formation of the particle-protein complexes can be described by a single exponential growth for all three investigated proteins, and the fitted pseudo-first-order rate constant increases linearly with particle concentration for each protein type. The derived second-order rate constant k varies with the protein hydrodynamic radius according to k∼RH(-4.70±0.01), indicating that the rate-limiting step at high particle concentrations is not the diffusional encounter between proteins and particles but rather the entry into the pores, consistent with the hydrodynamic radii of the three proteins being smaller but comparable to the pore radius of the particles. Copyright © 2015 Elsevier Inc. All rights reserved.

Measuring Sea-Ice Motion in the Arctic with Real Time Photogrammetry

NASA Astrophysics Data System (ADS)

Brozena, J. M.; Hagen, R. A.; Peters, M. F.; Liang, R.; Ball, D.

2014-12-01

The U.S. Naval Research Laboratory, in coordination with other groups, has been collecting sea-ice data in the Arctic off the north coast of Alaska with an airborne system employing a radar altimeter, LiDAR and a photogrammetric camera in an effort to obtain wide swaths of measurements coincident with Cryosat-2 footprints. Because the satellite tracks traverse areas of moving pack ice, precise real-time estimates of the ice motion are needed to fly a survey grid that will yield complete data coverage. This requirement led us to develop a method to find the ice motion from the aircraft during the survey. With the advent of real-time orthographic photogrammetric systems, we developed a system that measures the sea ice motion in-flight, and also permits post-process modeling of sea ice velocities to correct the positioning of radar and LiDAR data. For the 2013 and 2014 field seasons, we used this Real Time Ice Motion Estimation (RTIME) system to determine ice motion using Applanix's Inflight Ortho software with an Applanix DSS439 system. Operationally, a series of photos were taken in the survey area. The aircraft then turned around and took more photos along the same line several minutes later. Orthophotos were generated within minutes of collection and evaluated by custom software to find photo footprints and potential overlap. Overlapping photos were passed to the correlation software, which selects a series of "chips" in the first photo and looks for the best matches in the second photo. The correlation results are then passed to a density-based clustering algorithm to determine the offset of the photo pair. To investigate any systematic errors in the photogrammetry, we flew several flight lines over a fixed point on various headings, over an area of non-moving ice in 2013. The orthophotos were run through the correlation software to find any residual offsets, and run through additional software to measure chip positions and offsets relative to the aircraft

Design of a New Near-Infrared Ratiometric Fluorescent Nanoprobe for Real-Time Imaging of Superoxide Anions and Hydroxyl Radicals in Live Cells and in Situ Tracing of the Inflammation Process in Vivo.

PubMed

Liu, Rongjun; Zhang, Liangliang; Chen, Yunyun; Huang, Zirong; Huang, Yong; Zhao, Shulin

2018-04-03

The superoxide anion (O 2 •- ) and hydroxyl radical ( • OH) are important reactive oxygen species (ROS) used as biomarkers in physiological and pathological processes. ROS generation is closely related to the development of a variety of inflammatory diseases. However, the changes of ROS are difficult to ascertain with in situ tracing of the inflammation process by real-time monitoring, owing to the short half-lives of ROS and high tissue autofluorescence in vivo. Here we developed a new near-infrared (NIR) ratiometric fluorescence imaging approach by using a Förster resonance energy transfer (FRET)-based ratiometric fluorescent nanoprobe for real-time monitoring of O 2 •- and • OH generation and also by using in situ tracing of the inflammation process in vivo. The proposed nanoprobe was composed of PEG functionalized GQDs as the energy donor connecting to hydroIR783, serving as both the O 2 •- / • OH recognizing ligand and the energy acceptor. The nanoprobe not only exhibited a fast response to O 2 •- and • OH but also presented good biocomapatibility as well as a high photostability and signal-to-noise ratio. We have demonstrated that the proposed NIR ratiometric fluorescent nanoprobe can monitor the changes of O 2 •- and • OH in living RAW 264.7 cells via a drug mediating inflammation model and further realized visual monitoring of the change of O 2 •- and • OH in mice for in situ tracing of the inflammation process. Our design may provide a new paradigm for long-term and real-time imaging applications for in vivo tracing of the pathological process related to the inflammatory diseases.

Three-dimensional online surface reconstruction of augmented fluorescence lifetime maps using photometric stereo (Conference Presentation)

NASA Astrophysics Data System (ADS)

Unger, Jakob; Lagarto, Joao; Phipps, Jennifer; Ma, Dinglong; Bec, Julien; Sorger, Jonathan; Farwell, Gregory; Bold, Richard; Marcu, Laura

2017-02-01

Multi-Spectral Time-Resolved Fluorescence Spectroscopy (ms-TRFS) can provide label-free real-time feedback on tissue composition and pathology during surgical procedures by resolving the fluorescence decay dynamics of the tissue. Recently, an ms-TRFS system has been developed in our group, allowing for either point-spectroscopy fluorescence lifetime measurements or dynamic raster tissue scanning by merging a 450 nm aiming beam with the pulsed fluorescence excitation light in a single fiber collection. In order to facilitate an augmented real-time display of fluorescence decay parameters, the lifetime values are back projected to the white light video. The goal of this study is to develop a 3D real-time surface reconstruction aiming for a comprehensive visualization of the decay parameters and providing an enhanced navigation for the surgeon. Using a stereo camera setup, we use a combination of image feature matching and aiming beam stereo segmentation to establish a 3D surface model of the decay parameters. After camera calibration, texture-related features are extracted for both camera images and matched providing a rough estimation of the surface. During the raster scanning, the rough estimation is successively refined in real-time by tracking the aiming beam positions using an advanced segmentation algorithm. The method is evaluated for excised breast tissue specimens showing a high precision and running in real-time with approximately 20 frames per second. The proposed method shows promising potential for intraoperative navigation, i.e. tumor margin assessment. Furthermore, it provides the basis for registering the fluorescence lifetime maps to the tissue surface adapting it to possible tissue deformations.

Strain measurement of abdominal aortic aneurysm with real-time 3D ultrasound speckle tracking.

PubMed

Bihari, P; Shelke, A; Nwe, T H; Mularczyk, M; Nelson, K; Schmandra, T; Knez, P; Schmitz-Rixen, T

2013-04-01

Abdominal aortic aneurysm rupture is caused by mechanical vascular tissue failure. Although mechanical properties within the aneurysm vary, currently available ultrasound methods assess only one cross-sectional segment of the aorta. This study aims to establish real-time 3-dimensional (3D) speckle tracking ultrasound to explore local displacement and strain parameters of the whole abdominal aortic aneurysm. Validation was performed on a silicone aneurysm model, perfused in a pulsatile artificial circulatory system. Wall motion of the silicone model was measured simultaneously with a commercial real-time 3D speckle tracking ultrasound system and either with laser-scan micrometry or with video photogrammetry. After validation, 3D ultrasound data were collected from abdominal aortic aneurysms of five patients and displacement and strain parameters were analysed. Displacement parameters measured in vitro by 3D ultrasound and laser scan micrometer or video analysis were significantly correlated at pulse pressures between 40 and 80 mmHg. Strong local differences in displacement and strain were identified within the aortic aneurysms of patients. Local wall strain of the whole abdominal aortic aneurysm can be analysed in vivo with real-time 3D ultrasound speckle tracking imaging, offering the prospect of individual non-invasive rupture risk analysis of abdominal aortic aneurysms. Copyright © 2013 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

On the uncertainty in single molecule fluorescent lifetime and energy emission measurements

NASA Technical Reports Server (NTRS)

Brown, Emery N.; Zhang, Zhenhua; Mccollom, Alex D.

1995-01-01

Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least square methods agree and are optimal when the number of detected photons is large however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67% of those can be noise and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous poisson processes, we derive the exact joint arrival time probably density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. the ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background nose and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.

On the Uncertainty in Single Molecule Fluorescent Lifetime and Energy Emission Measurements

NASA Technical Reports Server (NTRS)

Brown, Emery N.; Zhang, Zhenhua; McCollom, Alex D.

1996-01-01

Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least squares methods agree and are optimal when the number of detected photons is large, however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67 percent of those can be noise, and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous Poisson processes, we derive the exact joint arrival time probability density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. The ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background noise and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.

Intestine pH measurements using fluorescence imaging: an in-vivo preliminary study

NASA Astrophysics Data System (ADS)

Marechal, Xavier-Marie; Mordon, Serge R.; Devoisselle, Jean-Marie; Begu, Sylvie; Mathieu, D.; Buys, Bruno; Dhelin, Guy; Lesage, Jean C.; Neviere, Remi; Chopin, Claude

1999-02-01

Measurement of gastrointestinal intramucosal pH has been recognized as an important factor in the detection of hypoxia-induced dysfunctions. However, current pH measurement techniques are limited in terms of time and spatial resolution. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-4,5- carboxyfluorescein (BCECF). This study aimed to demonstrate the feasibility of fluorescence imaging technique to measure in vivo the pH of intestine. The intestine was inserted in an optical chamber placed under a microscope. Animals were injected i.v. with the pH-sensitive fluorescent dye BCECF. Fluorescence was visualized by illuminating the intestine alternately at 490 and 470 nm. The emitted fluorescence was directed to an intensified camera. The ratio of emitted fluorescence at excitation wavelengths of 490 and 470 nm was measured, corrected and converted to pH by constructing a calibration curve. The pH controls were performed with a pH microelectrode correlated with venous blood gas sampling. We concluded that accurate pH measurements of rat intestine can be obtained by fluorescence imaging using BCECF. This technology could be easily adapted for endoscopic pH measurement.

Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; McGuinness, Dagmara; Faley, Shannon; Kolch, Walter; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines. PMID:19572560

Fluorescence-enhanced robotic radical prostatectomy using real-time lymphangiography and tissue marking with percutaneous injection of unconjugated indocyanine green: the initial clinical experience in 50 patients.

PubMed

Manny, Ted B; Patel, Manish; Hemal, Ashok K

2014-06-01

Pilot studies have demonstrated the utility of indocyanine green (ICG) sentinel lymphadenectomy for prostate cancer. Prior work has used ICG with radiocontrast agents injected at a separate procedure and relied on assistant-controlled fluorescence systems, making the technique costly and cumbersome. To describe the initial optimization and feasibility of fluorescence-enhanced robotic radical prostatectomy (FERRP) using real-time injection of ICG for tissue marking and identification of sentinel lymphatic drainage visualized by a fully integrated surgeon-controlled system. Patients with clinically localized prostate cancer at a tertiary referral center were offered FERRP. Ten patients participated in a pilot arm in which ICG dosing and injection technique were optimized. Fifty consecutive patients then underwent FERRP. After development of the space of Retzius, 0.4 ml of a 2.5 mg/ml ICG solution were injected into each lobe of the prostate using a robotically guided percutaneous needle. After ICG was allowed to travel through the pelvic lymphatics, lymphadenectomy was performed from the endopelvic fascia to the aortic bifurcation. Parameters describing the time course of tissue fluorescence and pelvic lymphangiography were systematically recorded. Lymphatic packets containing fluorescent nodes were considered sentinel. Percutaneous, robotic-guided ICG injection proved superior to cystoscope or transrectal delivery. Tissue marking was achieved in all patients, positively identifying the prostate with uniform fluorescence relative to the obturator nerve, seminal vesicles, vas deferens, and neurovascular pedicles at a mean time of 10 min postinjection. Sentinel nodes were identified in 76% of patients at a mean time of 30 min postinjection and had 100% sensitivity, 75.4% specificity, 14.6% positive predictive value, and 100% negative predictive value for the detection of nodal metastasis. FERRP is safe, feasible, and allows for reliable prostate tissue marking and

One step screening of retroviral producer clones by real time quantitative PCR.

PubMed

Towers, G J; Stockholm, D; Labrousse-Najburg, V; Carlier, F; Danos, O; Pagès, J C

1999-01-01

Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones.

Real-time data acquisition and control system for the measurement of motor and neural data

PubMed Central

Bryant, Christopher L.; Gandhi, Neeraj J.

2013-01-01

This paper outlines a powerful, yet flexible real-time data acquisition and control system for use in the triggering and measurement of both analog and digital events. Built using the LabVIEW development architecture (version 7.1) and freely available, this system provides precisely timed auditory and visual stimuli to a subject while recording analog data and timestamps of neural activity retrieved from a window discriminator. The system utilizes the most recent real-time (RT) technology in order to provide not only a guaranteed data acquisition rate of 1 kHz, but a much more difficult to achieve guaranteed system response time of 1 ms. The system interface is windows-based and easy to use, providing a host of configurable options for end-user customization. PMID:15698659

Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience.

PubMed

Kim, Sally A; Schwille, Petra

2003-10-01

Based on time-averaging fluctuation analysis of small fluorescent molecular ensembles in equilibrium, fluorescence correlation spectroscopy has recently been applied to investigate processes in the intracellular milieu. The exquisite sensitivity of fluorescence correlation spectroscopy provides access to a multitude of measurement parameters (rates of diffusion, local concentration, states of aggregation and molecular interactions) in real time with fast temporal and high spatial resolution. The introduction of dual-color cross-correlation, imaging, two-photon excitation, and coincidence analysis coupled with fluorescence correlation spectroscopy has expanded the utility of the technique to encompass a wide range of promising applications in living cells that may provide unprecedented insight into understanding the molecular mechanisms of intracellular neurobiological processes.

Real-time vibration measurement by a spatial phase-shifting technique with a tilted holographic interferogram.

PubMed

Nakadate, S; Isshiki, M

1997-01-01

Real-time vibration measurement by a tilted holographic interferogram is presented that utilizes the real-time digital fringe processor of a video signal. Three intensity data sampled at every one-third of the fringe spacing of the tilted fringes are used to calculate the modulation term of the fringe that is a function of a vibration amplitude. A three-dimensional lookup table performs the calculation in a TV repetition rate to give a new fringe profile that contours the vibration amplitude. Vibration modes at the resonant frequencies of a flat speaker were displayed on a monitor as changing the exciting frequency of vibration.

Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR▿

PubMed Central

Maeta, Kazuhiko; Ochi, Tomoya; Tokimoto, Keisuke; Shimomura, Norihiro; Maekawa, Nitaro; Kawaguchi, Nobuhisa; Nakaya, Makoto; Kitamoto, Yutaka; Aimi, Tadanori

2008-01-01

Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h. PMID:18378653

Real-time monitoring of the Trojan-horse effect of silver nanoparticles by using a genetically encoded fluorescent cell sensor.

PubMed

You, Fang; Tang, Wenqin; Yung, Lin-Yue Lanry

2018-04-26

Silver nanoparticles (AgNPs) are widely incorporated into commercial products due to their antimicrobial properties. As a consequence, concerns about the adverse effects induced by AgNPs to humans and the environment need to be carefully examined. The existing literature reveals that AgNPs exhibit certain toxic effects, but it remains to be proved whether AgNPs or the ionic silver (Ag+) released from AgNPs are the main toxic species. Here, a genetically encoded fluorescent protein sensor with high affinity to Ag+ was developed. The resulting sensor, MT2a-FRET, was found to be ratiometric, sensitive and selective toward only Ag+ but inert against AgNPs. This makes this sensor a potential useful tool for monitoring the real-time intracellular dissolutions of AgNPs. Our data supported that AgNPs display the "Trojan-horse" mechanism, where AgNPs are internalized by cells and undergo dissolution intracellularly. We further found that cells exhibited a detoxification ability to remove active Ag+ from cells in 48 hours.

Capture of Fluorescence Decay Times by Flow Cytometry

PubMed Central

Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

2012-01-01

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263

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.

Systematic characterization of maturation time of fluorescent proteins in living cells

PubMed Central

Balleza, Enrique; Kim, J. Mark; Cluzel, Philippe

2017-01-01

Slow maturation time of fluorescent proteins limits accurate measurement of rapid gene expression dynamics and effectively reduces fluorescence signal in growing cells. We used high-precision time-lapse microscopy to characterize, at two different temperatures in E. coli, the maturation kinetics of 50 FPs that span the visible spectrum. We identified fast-maturing FPs that yield the highest signal-to-noise ratio and temporal resolution in individual growing cells. PMID:29320486

Navigation surgery for intraoperative sentinel lymph node detection using Indocyanine green (ICG) fluorescence real-time imaging in breast cancer.

PubMed

Toh, U; Iwakuma, N; Mishima, M; Okabe, M; Nakagawa, S; Akagi, Y

2015-09-01

A new sensitive fluorescence imaging system was developed for the real-time identification of sentinel lymph nodes (SLNs) in patients with early breast cancer. The purpose of this study was to evaluate the utility of a color charge-coupled device camera system for the intraoperative detection of SLNs and to determine its clinical efficacy and sensitivity in patients with operable breast cancer. We assessed a total of 168 patients diagnosed with or suspected of having early-stage breast cancer without metastasis in SLNs. The intraoperative detection of SLNs was performed using the conventional Indigo Carmine dye (indigotindisulfonate sodium) technique combined with a new Indocyanine green (ICG) imaging system (HyperEye Medical System: HEMS, MIZUHO IKAKOGYO, Japan) to map SLNs, in which the lymphatic vessels and SLNs were visualized transcutaneously with illuminating ICG fluorescence. Between January 2012 and May 2013, SLNs were successfully identified in all 168 patients (detection rate: 100%). By histopathology, the sensitivity was 93.8% for the detection of the metastatic involvement of SLNs (15 of 16 nodal-positive patients). After a median follow-up of 30.5 months, none of the patients presented with axillary recurrence. These results suggest that the HEMS imaging system is a feasible and effective method for the detection of SLNs in breast cancer. Furthermore, the HEMS device permitted the transcutaneous visualization of lymphatic vessels under light conditions, thus facilitating the identification and detection of SLNs without affecting the surgical procedure, together with a high sensitivity and specificity.

Integrated instrument for dynamic light scattering and natural fluorescence measurements

NASA Astrophysics Data System (ADS)

Rovati, Luigi; Pollonini, Luca; Ansari, Rafat R.

2001-06-01

Over the past two decades, great efforts have been made in ophthalmology to use optical techniques based on dynamic light scattering and tissue natural fluorescence for early (at molecular level) diagnosis of ocular pathologies. In our previous studies, the relationship between the corneal AF and DLS decay widths of ocular tissues were established by performing measurements on diabetes mellitus patients. In those studies, corneal AF mean intensities were significantly correlated with DLS decay width measurements for each diabetic retinopathy grade in the vitreous and in the cornea. This suggested that the quality of the diagnosis could be significantly improved by properly combining these two powerful techniques into a single instrument. Our approach is based on modifying a commercial scanning ocular fluorometer (Fluorotron Master, Ocumetrics Inc., CA, USA) to include both techniques in the same scanning unit. This configuration provides both DLS and AF real time measurements from the same ocular volume: they can be located in each section of the optical axis of the eye from the cornea to the retina. In this paper, the optical setup of the new system is described and preliminary in-vitro and in-vivo measurements are presented.

Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence.

PubMed

Li, Dong; Zheng, Wei; Qu, Jianan Y

2008-10-15

A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.

Electrospray-assisted ultraviolet aerodynamic particle sizer spectrometer for real-time characterization of bacterial particles.

PubMed

Jung, Jae Hee; Lee, Jung Eun; Hwang, Gi Byoung; Lee, Byung Uk; Lee, Seung Bok; Jurng, Jong Soo; Bae, Gwi Nam

2010-01-15

The ultraviolet aerodynamic particle sizer (UVAPS) spectrometer is a novel, commercially available aerosol counter for real-time, continuous monitoring of viable bioaerosols based on the fluorescence induced from living microorganisms. For aerosolization of liquid-based microorganisms, general aerosolization methods such as atomization or nebulization may not be adequate for an accurate and quantitative characterization of the microorganisms because of the formation of agglomerated particles. In such cases, biological electrospray techniques have an advantage because they generate nonagglomerated particles, attributable to the repulsive electrical forces among particles with unipolar charges. Biological electrosprays are quickly gaining potential for the detection and control of living organisms in applications ranging from mass spectrometry to developmental microbiology. In this study, we investigated the size distribution, total concentration, and fluorescence percentage of bacterial particles in a real-time manner by electrospray-assisted UVAPS. A suspension containing Escherichia coli as a test microorganism was sprayed in a steady cone-jet mode using a specially designed electrospray system with a point-to-orifice-plate configuration based on charge-reduced electrospray size spectrometry. With the electrospray process, 98% of the total E. coli particle number concentration had a size of <1 mum and the geometric mean diameter was 0.779 mum, as compared with the respective values of 78% and 0.907 mum after nebulization. The fractions of fluorescence responsive particles and of particles that contained viable organisms in culture were 12% and 7%, respectively, from the electrospray process and 34% and 24% from nebulization. These results demonstrate that (1) the presence of agglomerated particles can lead to markedly overestimated fluorescence and culturability percentages compared with the values obtained from nonagglomerated particles, and (2) electrospray

Application of fiber Bragg grating sensors to real-time strain measurement of cryogenic tanks

NASA Astrophysics Data System (ADS)

Takeda, Nobuo; Mizutani, Tadahito; Hayashi, Kentaro; Okabe, Yoji

2003-08-01

Although many researches of strain measurement using fiber Bragg grating (FBG) sensors were conducted, there were few applications of FBG sensors to spacecraft in operation. It is very significant to develop an onboard system for the real-time strain measurement during the flight operation. In the present research, the real-time strain measurement of a composite liquid hydrogen (LH2) tank, which consisted of CFRP and aluminum liner, was attempted. Adhesive property of the FBG sensors was investigated first of all. As a result, UV coated FBG sensors and polyurethane adhesive were adopted. Then, reflection spectra from FBG sensors were measured through the tensile test at liquid helium (LHe) temperature. Since the center wavelength shifted in proportion to the applied strain, the FBG sensor was suitable as a precise strain sensor even at LHe temperature. Next, the development of an onboard FBG demodulator was discussed. This onboard demodulator was designed for weight saving to be mounted on a reusable rocket vehicle test (RVT) operated by the Institute of Space and Astronautical Science (ISAS). FBG sensors were bonded on the surface of the composite LH2 tank for the RVT. Then, strain measurement using the onboard demodulator was conducted through the cryogenic pressure test of the tank and compared with the result measured using the optical spectrum analyzer (OSA).

FPGA-based real-time phase measuring profilometry algorithm design and implementation

NASA Astrophysics Data System (ADS)

Zhan, Guomin; Tang, Hongwei; Zhong, Kai; Li, Zhongwei; Shi, Yusheng

2016-11-01

Phase measuring profilometry (PMP) has been widely used in many fields, like Computer Aided Verification (CAV), Flexible Manufacturing System (FMS) et al. High frame-rate (HFR) real-time vision-based feedback control will be a common demands in near future. However, the instruction time delay in the computer caused by numerous repetitive operations greatly limit the efficiency of data processing. FPGA has the advantages of pipeline architecture and parallel execution, and it fit for handling PMP algorithm. In this paper, we design a fully pipelined hardware architecture for PMP. The functions of hardware architecture includes rectification, phase calculation, phase shifting, and stereo matching. The experiment verified the performance of this method, and the factors that may influence the computation accuracy was analyzed.

A novel method for real-time skin impedance measurement during radiofrequency skin tightening treatments.

PubMed

Harth, Yoram; Lischinsky, Daniel

2011-03-01

The thermal effects of monopolar and bipolar radiofrequency (RF) have been proven to be beneficial in skin tightening. Nevertheless, these effects were frequently partial or unpredictable because of the uncontrolled nature of monopolar or unipolar RF and the superficial nature of energy flow for bipolar or tripolar configurations. One of the hypotheses for lack or predictability of efficacy of the first-generation RF therapy skin tightening systems is lack of adaptation of delivered power to differences in individual skin impedance. A novel multisource phase-controlled system was used (1 MHz, power range 0-65 W) for treatment and real-time skin impedance measurements in 24 patients (EndyMed PRO™; EndyMed, Cesarea, Israel). This system allows continuous real-time measurement of skin impedance delivering constant energy to the patient skin independent of changes in its impedance. More than 6000 unique skin impedance measurements on 22 patients showed an average session impedance range was 215-584 Ohm with an average of 369 Ohm (standard deviation of 49 Ohm). Analyzing individual pulses (total of 600 readings) showed a significant decrease in impedance during the pulse. These findings validate the expected differences in skin impedance between individual patients and in the same patients during the treatment pulse. Clinical study on 30 patients with facial skin aging using the device has shown high predictability of efficacy (86.7% of patients had good results or better at 3 months' follow-up [decrease of 2 or more grades in Fitzpatrick's wrinkle scale]). The real-time customization of energy according to skin impedance allows a significantly more accurate and safe method of nonablative skin tightening with more consistent and predictable results. © 2011 Wiley Periodicals, Inc.

Fluorescence Microscopy of Single Molecules

ERIC Educational Resources Information Center

Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

2004-01-01

The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

Real-Time H2 O2 Measurements in Bone Marrow Mesenchymal Stem Cells (MSCs) Show Increased Antioxidant Capacity in Cells From Osteoporotic Women.

PubMed

Román, Flavia; Urra, Carla; Porras, Omar; Pino, Ana María; Rosen, Clifford J; Rodríguez, Juan Pablo

2017-03-01

Oxidative stress (OS) derived from an increase in intracellular reactive oxygen species (ROS) is a major determinant of aging and lifespan. It has also been associated with several age-related disorders, like postmenopausal osteoporosis of Mesenchymal stem cells (MSCs). MSCs are the common precursors for osteoblasts and adipocytes; appropriate commitment and differentiation of MSCs into a specific phenotype is modulated, among other factors, by ROS balance. MSCs have shown more resistance to ROS than differentiated cells, and their redox status depends on complex and abundant anti-oxidant mechanisms. The purpose of this work was to analyze in real time, H 2 O 2 signaling in individual h-MSCs, and to compare the kinetic parameters of H 2 O 2 management by cells derived from both control (c-) and osteoporotic (o-) women. For these purposes, cells were infected with a genetically encoded fluorescent biosensor named HyPer, which is specific for detecting H 2 O 2 inside living cells. Subsequently, cells were sequentially challenged with 50 and 500 μM H 2 O 2 pulses, and the cellular response was recorded in real time. The results demonstrated adequate expression of the biosensor allowing registering fluorescence from HyPer at a single cell level. Comparison of the response of c- and o-MSCs to the oxidant challenges demonstrated improved antioxidant activity in o-MSCs. This was further corroborated by measuring the relative expression of mRNAs for catalase, superoxide dismutase-1, thioredoxine, and peroxiredoxine, as well as by cell-surviving capacity under short-term H 2 O 2 treatment. We conclude that functional differences exist between healthy and osteoporotic human MSCs. The mechanism for these differences requires further study. J. Cell. Biochem. 118: 585-593, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A novel real time imaging platform to quantify macrophage phagocytosis.

PubMed

Kapellos, Theodore S; Taylor, Lewis; Lee, Heyne; Cowley, Sally A; James, William S; Iqbal, Asif J; Greaves, David R

2016-09-15

Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM® real time imaging platform for macrophage phagocytosis based on pHrodo® pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyte™ Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20× less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Real-time imaging of nitric oxide production in living cells with 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacence by invert fluorescence microscope.

PubMed

Huang, Ke-Jing; Wang, Hong; Ma, Ming; Zhang, Xian; Zhang, Hua-Shan

2007-02-01

Although the importance of nitric oxide (NO) as a signalling molecule in many biological processes is becoming increasingly evident, many proposed and potential biological functions of NO still remain unclear. Bioimaging is a good technique to visualize observation of nitric oxide in biological samples. In this report, a fluorescent probe, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABODIPY), has been first applied to real-time image NO produced in PC12 cells, Sf9 cells and human vascular endothelial cells at the presence of l-arginine with inverted fluorescence microscope. NO production in the cells is successfully captured and imaged with fine temporal and spatial resolution. The results prove that the probe combined with inverted fluorescence microscope can be developed into a sensitive and selective method for further study of NO release from cells.

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)

A real-time spectrum acquisition system design based on quantum dots-quantum well detector

NASA Astrophysics Data System (ADS)

Zhang, S. H.; Guo, F. M.

2016-01-01

In this paper, we studied the structure characteristics of quantum dots-quantum well photodetector with response wavelength range from 400 nm to 1000 nm. It has the characteristics of high sensitivity, low dark current and the high conductance gain. According to the properties of the quantum dots-quantum well photodetectors, we designed a new type of capacitive transimpedence amplifier (CTIA) readout circuit structure with the advantages of adjustable gain, wide bandwidth and high driving ability. We have implemented the chip packaging between CTIA-CDS structure readout circuit and quantum dots detector and tested the readout response characteristics. According to the timing signals requirements of our readout circuit, we designed a real-time spectral data acquisition system based on FPGA and ARM. Parallel processing mode of programmable devices makes the system has high sensitivity and high transmission rate. In addition, we realized blind pixel compensation and smoothing filter algorithm processing to the real time spectrum data by using C++. Through the fluorescence spectrum measurement of carbon quantum dots and the signal acquisition system and computer software system to realize the collection of the spectrum signal processing and analysis, we verified the excellent characteristics of detector. It meets the design requirements of quantum dot spectrum acquisition system with the characteristics of short integration time, real-time and portability.

PERTS: A Prototyping Environment for Real-Time Systems

NASA Technical Reports Server (NTRS)

Liu, Jane W. S.; Lin, Kwei-Jay; Liu, C. L.

1993-01-01

PERTS is a prototyping environment for real-time systems. It is being built incrementally and will contain basic building blocks of operating systems for time-critical applications, tools, and performance models for the analysis, evaluation and measurement of real-time systems and a simulation/emulation environment. It is designed to support the use and evaluation of new design approaches, experimentations with alternative system building blocks, and the analysis and performance profiling of prototype real-time systems.

Automated high-throughput flow-through real-time diagnostic system

DOEpatents

Regan, John Frederick

2012-10-30

An automated real-time flow-through system capable of processing multiple samples in an asynchronous, simultaneous, and parallel fashion for nucleic acid extraction and purification, followed by assay assembly, genetic amplification, multiplex detection, analysis, and decontamination. The system is able to hold and access an unlimited number of fluorescent reagents that may be used to screen samples for the presence of specific sequences. The apparatus works by associating extracted and purified sample with a series of reagent plugs that have been formed in a flow channel and delivered to a flow-through real-time amplification detector that has a multiplicity of optical windows, to which the sample-reagent plugs are placed in an operative position. The diagnostic apparatus includes sample multi-position valves, a master sample multi-position valve, a master reagent multi-position valve, reagent multi-position valves, and an optical amplification/detection system.

Real-time quasi-3D tomographic reconstruction

NASA Astrophysics Data System (ADS)

Buurlage, Jan-Willem; Kohr, Holger; Palenstijn, Willem Jan; Joost Batenburg, K.

2018-06-01

Developments in acquisition technology and a growing need for time-resolved experiments pose great computational challenges in tomography. In addition, access to reconstructions in real time is a highly demanded feature but has so far been out of reach. We show that by exploiting the mathematical properties of filtered backprojection-type methods, having access to real-time reconstructions of arbitrarily oriented slices becomes feasible. Furthermore, we present , software for visualization and on-demand reconstruction of slices. A user of can interactively shift and rotate slices in a GUI, while the software updates the slice in real time. For certain use cases, the possibility to study arbitrarily oriented slices in real time directly from the measured data provides sufficient visual and quantitative insight. Two such applications are discussed in this article.

Initial formal toxicity evaluation of APC-2, a novel fluorescent tracer agent for real-time measurement of glomerular filtration rate in preparation for a first-in-man clinical trial

NASA Astrophysics Data System (ADS)

Bugaj, Joseph E.; Dorshow, Richard B.

2014-03-01

The fluorescent tracer agent 2,5-bis[N-(1-carboxy-2-hydroxy)]carbamoyl-3,6-diaminopyrazine, designated APC-2, has been developed with properties and attributes necessary for use as a direct measure of glomerular filtration rate (GFR). Comparison to known standard exogenous GFR agents in animal models has demonstrated an excellent correlation. A clinical trial to demonstrate this same correlation in humans is in preparation. A battery of formal toxicity tests necessary for regulatory clearance to proceed with a clinical trial has been recently completed on this new fluorescent tracer agent. These include single dose toxicity studies in rats and dogs to determine overall toxicity and toxicokinetics of the compound. Blood compatibility, mutation assay, chromosomal aberration assay, and several other assays were also completed. Toxicity assessments were based on mortality, clinical signs, body weight, food consumption and anatomical pathology. Blood samples were collected to assess pharmacokinetic parameters including half-life, area under the curve, and clearance. Urine samples were collected to assess distribution. Doses of up to 200-300 times the estimated human dose were administered. No test-article related effects were noted on body weight, food consumption, ophthalmic observations and no abnormal pathology was seen in either macroscopic or microscopic evaluations of any organs or tissues. All animals survived to scheduled sacrifice. Transient discoloration of skin and urine was noted at the higher dose levels in both species as expected from a highly fluorescent compound and was not considered pathological. Thus initial toxicology studies of this new fluorescent tracer agent APC-2 have resulted in no demonstrable pathological test article concerns.

Novel techniques of real-time blood flow and functional mapping: technical note.

PubMed

Kamada, Kyousuke; Ogawa, Hiroshi; Saito, Masato; Tamura, Yukie; Anei, Ryogo; Kapeller, Christoph; Hayashi, Hideaki; Prueckl, Robert; Guger, Christoph

2014-01-01

There are two main approaches to intraoperative monitoring in neurosurgery. One approach is related to fluorescent phenomena and the other is related to oscillatory neuronal activity. We developed novel techniques to visualize blood flow (BF) conditions in real time, based on indocyanine green videography (ICG-VG) and the electrophysiological phenomenon of high gamma activity (HGA). We investigated the use of ICG-VG in four patients with moyamoya disease and two with arteriovenous malformation (AVM), and we investigated the use of real-time HGA mapping in four patients with brain tumors who underwent lesion resection with awake craniotomy. Real-time data processing of ICG-VG was based on perfusion imaging, which generated parameters including arrival time (AT), mean transit time (MTT), and BF of brain surface vessels. During awake craniotomy, we analyzed the frequency components of brain oscillation and performed real-time HGA mapping to identify functional areas. Processed results were projected on a wireless monitor linked to the operating microscope. After revascularization for moyamoya disease, AT and BF were significantly shortened and increased, respectively, suggesting hyperperfusion. Real-time fusion images on the wireless monitor provided anatomical, BF, and functional information simultaneously, and allowed the resection of AVMs under the microscope. Real-time HGA mapping during awake craniotomy rapidly indicated the eloquent areas of motor and language function and significantly shortened the operation time. These novel techniques, which we introduced might improve the reliability of intraoperative monitoring and enable the development of rational and objective surgical strategies.

Redox-initiated hydrogel system for detection and real-time imaging of cellulolytic enzyme activity.

PubMed

Malinowska, Klara H; Verdorfer, Tobias; Meinhold, Aylin; Milles, Lukas F; Funk, Victor; Gaub, Hermann E; Nash, Michael A

2014-10-01

Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of real-time instruments and gravimetric method when measuring particulate matter in a residential building.

PubMed

Wang, Zuocheng; Calderón, Leonardo; Patton, Allison P; Sorensen Allacci, MaryAnn; Senick, Jennifer; Wener, Richard; Andrews, Clinton J; Mainelis, Gediminas

2016-11-01

This study used several real-time and filter-based aerosol instruments to measure PM 2.5 levels in a high-rise residential green building in the Northeastern US and compared performance of those instruments. PM 2.5 24-hr average concentrations were determined using a Personal Modular Impactor (PMI) with 2.5 µm cut (SKC Inc., Eighty Four, PA) and a direct reading pDR-1500 (Thermo Scientific, Franklin, MA) as well as its filter. 1-hr average PM 2.5 concentrations were measured in the same apartments with an Aerotrak Optical Particle Counter (OPC) (model 8220, TSI, Inc., Shoreview, MN) and a DustTrak DRX mass monitor (model 8534, TSI, Inc., Shoreview, MN). OPC and DRX measurements were compared with concurrent 1-hr mass concentration from the pDR-1500. The pDR-1500 direct reading showed approximately 40% higher particle mass concentration compared to its own filter (n = 41), and 25% higher PM 2.5 mass concentration compared to the PMI 2.5 filter. The pDR-1500 direct reading and PMI 2.5 in non-smoking homes (self-reported) were not significantly different (n = 10, R 2 = 0.937), while the difference between measurements for smoking homes was 44% (n = 31, R 2 = 0.773). Both OPC and DRX data had substantial and significant systematic and proportional biases compared with pDR-1500 readings. However, these methods were highly correlated: R 2 = 0.936 for OPC versus pDR-1500 reading and R 2 = 0.863 for DRX versus pDR-1500 reading. The data suggest that accuracy of aerosol mass concentrations from direct-reading instruments in indoor environments depends on the instrument, and that correction factors can be used to reduce biases of these real-time monitors in residential green buildings with similar aerosol properties. This study used several real-time and filter-based aerosol instruments to measure PM 2.5 levels in a high-rise residential green building in the northeastern United States and compared performance of those instruments. The data show that while the use of real-time

Real-Time Unsteady Loads Measurements Using Hot-Film Sensors

NASA Technical Reports Server (NTRS)

Mangalam, Arun S.; Moes, Timothy R.

2004-01-01

Several flight-critical aerodynamic problems such as buffet, flutter, stall, and wing rock are strongly affected or caused by abrupt changes in unsteady aerodynamic loads and moments. Advanced sensing and flow diagnostic techniques have made possible simultaneous identification and tracking, in realtime, of the critical surface, viscosity-related aerodynamic phenomena under both steady and unsteady flight conditions. The wind tunnel study reported here correlates surface hot-film measurements of leading edge stagnation point and separation point, with unsteady aerodynamic loads on a NACA 0015 airfoil. Lift predicted from the correlation model matches lift obtained from pressure sensors for an airfoil undergoing harmonic pitchup and pitchdown motions. An analytical model was developed that demonstrates expected stall trends for pitchup and pitchdown motions. This report demonstrates an ability to obtain unsteady aerodynamic loads in real time, which could lead to advances in air vehicle safety, performance, ride-quality, control, and health management.

Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics.

PubMed

Wei, Liping; Yan, Wenrong; Ho, Derek

2017-12-04

Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices.

Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics

PubMed Central

Yan, Wenrong; Ho, Derek

2017-01-01

Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices. PMID:29207568

Evaluation of the ACEC Benchmark Suite for Real-Time Applications

DTIC Science & Technology

1990-07-23

1.0 benchmark suite waSanalyzed with respect to its measuring of Ada real-time features such as tasking, memory management, input/output, scheduling...and delay statement, Chapter 13 features , pragmas, interrupt handling, subprogram overhead, numeric computations etc. For most of the features that...meant for programming real-time systems. The ACEC benchmarks have been analyzed extensively with respect to their measuring of Ada real-time features

[Identification of human pathogenic variola and monkeypox viruses by real-time polymerase chain reaction].

PubMed

Kostina, E V; Gavrilova, E V; Riabinin, V A; Shchelkunov, S N; Siniakov, A N

2009-01-01

A kit of specific oligonucleotide primers and hybridization probes has been proposed to detect orthopoxviruses (OPV) and to discriminate human pathogenic viruses, such as variola virus and monkey virus by real-time polymerase chain reaction (PCR). For real-time PCR, the following pairs of fluorophore and a fluorescence quencher were used: TAMRA-BHQ2 for genus-specific probes and FAM-BHQ1 for species-specific ones (variola virus, monkeypox virus, ectomelia virus). The specificity of this assay was tested on 38 strains of 6 OPV species and it was 100%.

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

PubMed

Yang, Yu; Bai, Lin; Hu, Kong-Xin; Yang, Zhi-Hong; Hu, Jian-Ping; Wang, Jing

2012-08-01

Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

Real-Time Visualization of Tissue Ischemia

NASA Technical Reports Server (NTRS)

Bearman, Gregory H. (Inventor); Chrien, Thomas D. (Inventor); Eastwood, Michael L. (Inventor)

2000-01-01

A real-time display of tissue ischemia which comprises three CCD video cameras, each with a narrow bandwidth filter at the correct wavelength is discussed. The cameras simultaneously view an area of tissue suspected of having ischemic areas through beamsplitters. The output from each camera is adjusted to give the correct signal intensity for combining with, the others into an image for display. If necessary a digital signal processor (DSP) can implement algorithms for image enhancement prior to display. Current DSP engines are fast enough to give real-time display. Measurement at three, wavelengths, combined into a real-time Red-Green-Blue (RGB) video display with a digital signal processing (DSP) board to implement image algorithms, provides direct visualization of ischemic areas.

Fluorescence quantum yield measurements of fluorescent proteins: a laboratory experiment for a biochemistry or molecular biophysics laboratory course.

PubMed

Wall, Kathryn P; Dillon, Rebecca; Knowles, Michelle K

2015-01-01

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper-level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum. © 2014 The International Union of Biochemistry and Molecular Biology.