Efficient detection of single DNA fragments in flowing sample streams by two-photon fluorescence excitation

This paper reports the demonstration of efficient single molecule detection in flow cytometry by two-photon fluorescence excitation. We have used two-photon excitation (TPE) to detect single DNA fragments as small as 383 base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNA complexes was accomplished using a mode-locked, 120 fs pulse width Ti:sapphire laser operating at 810 nm. POPO-1 labeled DNA fragments of 1.1 kilobase pairs (kbp) and larger were sequentially detected in our flow cytometry system with a detection efficiency of nearly 100%. The detection efficiency for the 383 bp DNA fragments was approximately 75%. We also demonstrate the ability to distinguish between different sized DNA fragments in a mixture by their individual fluorescence burst sizes by TPE. These studies indicate that using TPE for single molecule flow cytometry experiments lowers the intensity of the background radiation by approximately an order of magnitude compared to one-photon excitation, due to the large separation between the excitation and emission wavelengths in TPE.     

Single-molecule identification by spectrally and time-resolved fluorescence detection

A method to identify single molecules rapidly and with high efficiency based on simple probability considerations is proposed. In principle, any property of a detected photon in a single-molecule fluorescence experiment, e.g., emission wavelength, arrival time after pulsed excitation, and polarization, can be analyzed within the framework of the outlined methodology. Monte Carlo simulations show that less than 500 photons are needed to assign an observed single molecule to one out of four species with a confidence level higher than 99.9%. We show that single dye molecules of four different dyes embedded in a polymer film can be identified with time-correlated single-photon counting spectrally resolved in two channels.     

Single-molecule detection and identification of multiple species by multiparameter fluorescence detection

Two general strategies are introduced to identify and quantify single molecules in dilute solutions by employing a spectroscopic method for data registration and specific burst analysis, denoted multiparameter fluorescence detection (MFD). MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time and allows for selection of specific events for subsequent analysis. Using the multiple fluorescence dimensions, we demonstrate a dye labeling scheme of oligonucleotides, by which it is possible to identify and separate 16 different compounds in the mixture via their characteristic pattern by MFD. Such identification procedures and multiplex assays with single-molecule sensitivity may have a great impact on screening of species and events that do not lend themselves so easily to amplification, such as disease-specific proteins and their interactions.     

Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami

DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with <30 nm resolution. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.     

Reducing sample size by tightening test conditions

The inspection of measurement devices according to statistical sampling plans allows conclusions to be drawn about the reliability of a whole population of devices. However, confirming high reliability levels requires large sample sizes and is thus expensive or even infeasible. For example, a reliability of 99.5% can only be guaranteed with 90% confidence by inspecting each item in a population of 280 (see ISO 2859‐2). When reliability is judged by not exceeding a certain threshold, this research provides a convenient solution allowing considerably more efficient sampling plans. Under certain distributional assumptions, in particular, we have proved that if 100q% of a population meets a tighter threshold Δ/γ, then at least 100p% of the population meets threshold Δ(with p>q, γ>1). The importance and effect of different distributional assumptions are demonstrated and relevant scenarios for the parameters (p,q,γ) presented. Verifying that a smaller portion of devices comply requires smaller sample sizes. Costs may thus decrease when more stringent specifications are verified. For example, up to 98% of utility meters in Germany are required to measure correctly at inspections, to ensure a reliability of 95% in the future. Instead of applying costly sampling plans to meters in use to demonstrate these high reliability levels, this research enables the sample size to be reduced, eg, by half.     

Single-molecule analysis and lifetime estimates of heterogeneous low-count-rate time-correlated fluorescence data

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.     

Treatment of hazardous materials spills in flowing streams with floating mass transfer agents

Field studies were conducted in a Flowing Stream Test Facility to determine parameters governing application of floating media (in this case, commercially-available activated carbon) to flowing streams for the purpose of treating hazardous materials spills in situ. It was found that removal efficiency is highly dependent on prompt location of the contaminant plume, even dispersion of media over the water surface, and favorable environmental conditions. This technique is most effective for concentrated plumes in small streams, and removal efficiencies increase as the size of the spill increases. Removal efficiencies ranged from 50 to > 95%, depending upon the substance being used in the tests.Further investigation should focus on ballast and packaging techniques, methods for increasing efficiency of contact, prevention of carbon buildup along stream banks, and efficiency of spent carbon collection.     

Single-molecule fluorescence resonance energy transfer in nanopipets: improving distance resolution and concentration range

In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of approximately 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (<10(9) M-1) using minute sample amounts (<5 microL). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.     

Single-molecule DNA patterning and detection by padlock probing and rolling circle amplification in microchannels for analysis of small sample volumes

The rolling circle amplification (RCA) is a versatile DNA amplification method in which a DNA molecule is amplified using a single DNA primer, allowing the product to be counted as a single dot. Circular templates for RCA can arise from padlock probes in highly specific DNA target-mediated ligation reactions. However, improvement of detection efficiency represents an important challenge. In homogeneous assays, the detection efficiency is generally only under 0.1%, mainly because the sample volume is too large compared with the detection volume. Here, we used microchannel surfaces in a glass microchip for DNA detection in small volume samples. First, DNA patterning on glass surfaces in microchannels was demonstrated using chemical surface patterning by UV light. By using a photochemical reaction, we realized DNA patterning in a closed space. Second, RCA was demonstrated using dilutions of target molecules, and a calibration curve was obtained. The highest detection efficiency was 22.5% by virtue of the reduced sample volumes from several hundred microliters to 5.0 nL. Accordingly, a countable number of DNA molecules was successfully detected. This method is suitable for analysis of very small volume samples such as single cells, especially by using extended-nanochannels with dimensions of 10-1000 nm.     

Airflow rate in the quantitation of volatiles in air streams by solid-phase microextraction

A previously reported method for nonequilibrium quantitation of air-borne volatiles from air streams by solid-phase microextraction (SPME) was improved by broadening its scope. The original method was defined for the 100-microm poly(dimethylsiloxane) fiber type for a wide range of analytes, sampling temperatures, and sampling times, but only for four specific airflow configurations. The present study extends the choice of volumetric airflow rates to a continuous range between 2 and 220 mL/min. Kinetics of absorption was characterized for 21 different airflow rates within this range using n-alkanes of 11-18 carbons. Nonlinear regression analysis was used to develop a relationship between airflow rate and absorption kinetics and then to integrate these results into the previous model. The overall model (with 8 fitted degrees of freedom and based on 2240 measurements) had an r2 value of 0.9972 and residual variability (RSD) of 9.75%, which compared favorably with the sampling precision of SPME (approximately 5%). The method allows absolute quantitation by SPME for a broad range of analytes and sampling parameters without prior calibration of the individual fiber and regardless of whether equilibration is complete. Simulations are presented that demonstrate how the choice of airflow rate can affect quantitation.     

Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis.

A method has been developed for detecting DNA separated by capillary gel electrophoresis (CGE) using single molecule photon burst counting. A confocal fluorescence microscope was used to observe the fluorescence bursts from single molecules of DNA multiply labeled with the thiazole orange derivative T06 as they passed through the approximately 2 micrometer diameter focused laser beam. Amplified photoelectron pulses from the photomultiplier are grouped into bins of 360-450 micros in duration, and the resulting histogram is stored in a computer for analysis. Solutions of M13 DNA were first flowed through the capillary at various concentrations, and the resulting data were used to optimize the parameters for digital filtering using a low-pass Fourier filter, selecting a discriminator level for peak detection, and applying a peak-calling algorithm. Statistical analyses showed that (i) the number of M13 molecules counted versus concentration was linear with slope = 1, (ii) the average burst duration was consistent with the expected transit time of a single molecule through the laser beam, and (iii) the number of detected molecules was consistent with single molecule detection. The optimized single molecule counting method was then applied to an electrophoretic separation of M13 DNA and to a separation of pBR 322 DNA from pRL 277 DNA. Clusters of discreet fluorescence bursts were observed at the expected appearance time of each DNA band. The autocorrelation function of these data indicated transit times that were consistent with the observed electrophoretic velocity. These separations were easily detected when only 50-100 molecules of DNA per band traveled through the detection region. This new detection technology should lead to the routine analysis of DNA in capillary columns with an on-column sensitivity of approximately 100 DNA molecules/band or better.     

Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy

Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam.     

熔融制样X射线荧光光谱法测定矿石中五氧化二钒的含量

使用熔融制样-X射线荧光光谱法(XRF),以四硼酸锂做熔剂,样品与熔剂比为1∶10熔融制样.分析矿石中0.006％～98％的五氧化二钒含量.目前五氧化二钒标准物质较少,不能满足各含量段矿石类样品的准确定量要求.本方法通过加入高纯V2O5制备人工标准样品和现有五氧化二钒标准物质相结合绘制工作曲线,解决了现有标准物质较少的...     

Confidence intervals and sample size determination for Cpm

The capability index, C_pm, sometimes called the Taguchi index, has the desirable characteristic of being sensitive to both dispersion and deviation of the process average from the engineering target. As a result, the proposed estimators of C_pm have a sampling distribution that is dependent on the non‐central chi‐square distribution. Hence, constructing confidence intervals, performing hypothesis testing or estimating sample size requirements necessitates manipulation of a rather complex functional expression, typically beyond the capabilities of practitioners who need readily available tools. Here, a simple graphical procedure is proposed and illustrated for obtaining exact confidence intervals for C_pm. The graphical procedure allows the user to simply enter the graph with an estimate of the index and a value of the non‐centrality parameter for a given sample size to arrive at end‐points of 90%, 95% or 99% one‐sided or two‐sided confidence intervals. Detailed tables are also provided to assist the user for a wider range of sample values and sample sizes. In addition, a procedure is also presented for determining the minimum sample size required for attaining a pre‐specified level of accuracy of the C_pm. Extensive tables are provided for the user with a simple example illustrating the facility of the technique. Copyright © 2001 John Wiley & Sons, Ltd.     

Relationship of chlorine decay and THMs formation to NOM size

Because of increasing concern about balancing health risks for pathogen control and disinfection by-product (DBP) formation in water supplies, utilities are forced to closely examine and optimize their disinfection practices. A better understanding of the relationship between the molecular weights of the natural organic matter (NOM), chlorine decay kinetics and THMs formation can help the utilities to minimize the DBP concentrations, providing healthier and microbially safer water. The authors present data on chlorine decay kinetics and total trihalomethanes (TTHM) formation kinetics and modeling with different molecular weights NOM fractions of Mississippi River water. TTHM modeling results indicated that the TTHM formation in fractionated NOM was a function of chlorine consumption. TTHM yield coefficients ranged from 31 to 42 microg-TTHM/mg-Cl2. As the molecular weight of the fractions decreased, TTHM yield coefficients increased.     

原子荧光法测定生态地球化学调查土壤样品中的砷、汞

研究了原子荧光法测定生态地球化学调查样品中的砷、汞的测量条件。As、Hg方法的检出限分别为0.2mg/kg和0.0042mg/kg。As的相对标准偏差(RSD)为≤4.10%,Hg相对标准偏差(RSD)为≤6.38%,方法的各项指标完全满足生态地球化学调查样品检测的要求。