Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed. Correspondence to: R. Rigler     

Dye exclusion artifact in flow cytometers

BACKGROUND: Cells exclude their own volume of dye solution in the sample flow which carries them through the flow chamber of the flow cytometer, thereby affecting the otherwise constant signal arising from the fluorescence of this solution. Under certain conditions, this phenomenon may significantly influence the fluorescence signal of the cells. MATERIALS AND METHODS: Using the slit scan technique, we studied this phenomenon as observed for monodisperse polystyrene particles in fluorescein solution. RESULTS: The measurements show that dye solution accumulates just in front of the particle and just behind it, with a relative void in between. This phenomenon is most likely caused by the rapid constriction of the flow as it enters the orifice of the nozzle or flow chamber, giving rise to a pulse of fluorescence which adds to that of the particle or cell itself. The magnitude of this artifact depends on the design and dimensions of the nozzle/flow chamber as well as on the rate of sample flow. CONCLUSIONS: The dye exclusion artifact may affect measurements of cells when they are in a dye solution having a fluorescence per unit volume which is significant compared to that of the cells, especially at low sample flow rates.     

在荧光漂白恢复测量中荧光恢复起始时间的研究

在荧光漂白恢复测量中,漂白过程结束的准确时间相对于漂白控制信号后沿会有一定的延迟时间,不易确定.光电倍增管快门开启又要推迟一段时间.因此,荧光恢复起始时间,即漂白结束的瞬间不能准确给出,开始阶段数据收集不全.本文分析了荧光恢复起始时间不准确所造成的误差,测量了起始时间.并尽量减少此误差,使恢复初期收集到的数据更完全,荧光恢复过程的时间座标更准确.     

解离增强镧系元素荧光免疫分析灵敏度的改进

为提高解离增强镧系荧光免疫分析(DELFIA)的灵敏度或信／噪比,进行了一些重要的方法学研究．观察到了对于不同的铕量,荧光响应和信／噪比都随着增强液体积而明显地变化．对于确定的铕量,存在一个最佳体积,且铕量越小,其最佳体积也越小．实验中选择最佳体积是重要的．研究了增强液制备技术,发展了微滴定板条有效的清洗和干燥方法,使本底荧光明显降低．     

Fluorescence correlation spectroscopy in the nanosecond time range: Photon antibunching in dye fluorescence

Possibilities for the use of fluorescence correlation spectroscopy in the nanosecond time range are demonstrated. The experiment is based on a cw argon ion laser, a microfluorimeter, two photon detectors, and a time-to-analog converting system. Experiments using solutions of rhodamine 6G and pyronine G in water at concentrations of about 20 molecules per sample volume are reported. The photon anticorrelation component decaying with a time constant close to the excited state lifetime was observed.     

应用小波变换检测细胞显微荧光图像中的囊泡

文章提出了一种用小波变换来检测生物荧光图像中囊泡的方法。作者用à trous小波对图像进行小波变换,然后求出每层系数的中值绝对偏差σ,并用t=kσ/0.67作为阈值对每层系数进行门限滤波,然后通过提取小波变换系数来重构图像。通过设计实验与常用的“rolling ball”算法对比,发现小波变换算法在低信噪比的情况下,具有更好的灵敏度;对于形状大小不同的信号,具有更好的稳定性;而且对于信号的细节信息具有更好的保真性。     

Determination of axonal transport velocities via image cross- and autocorrelation

On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. O. Welzel and D. Boening contributed equally to this work.     

Generalization of the variance-covariance method for microdosimetric measurements

Numerical simulations in terms of microdosimetric data for 5-MeV neutrons are utilized to test the twin detector method of microdosimetric measurements in its new, extended form. Three different conditions of measurement are considered: (I) constant dose increments per measurement, (II) fluctuating dose increments per measurement, and (III) fluctuating dose increments combined with slow changes of the dose-rate ratio for the two detectors. Under each of the conditions, large numbers of measurement series are simulated, and the estimation formulae for the mean and the two subsequent moments of the dose-weighted single-event distribution, d(y), are applied. The estimation formulae exist in three different forms: uncorrected formulae that apply to condition I, corrected formulae that are valid also for condition II, and fully corrected formulae that remain applicable under condition III. The results of the simulations indicate the acceptable ranges of doses per measurement interval. It can be seen that all three parameters are obtainable through the twin detector method, and that the fully corrected formulae for the two lower moments are generally applicable. The determination of the third moment is possible only under limited conditions, and the fully corrected formula is not useful in this case, while the corrected formula has some applicability even under condition III. Received: 10 January 1996 / Accepted in revised form: 27 May 1996     

High-resolution alignment of action potential waveforms using cubic spline interpolation

A cubic spline interpolation technique is applied to the problem of aligning action potential waveforms. Interpolation is an attractive alternative to sampling at many times the Nyquist rate in order to reduce errors caused by asynchronous sampling of rapidly changing waveforms. Alignment is achieved by locating the peak of the interpolated waveform, which can be found by solving a quadratic equation. The waveform is then reconstructed for comparison with existing templates. The technique was tested using simulated noisy, randomly arriving waveforms, the interpolated signal and alignment time errors being computed as functions of the signal/noise ratio. The spline technique is superior in accuracy to sampling at eight-times the Nyquist rate and is comparable to a Fourier-transform-based interpolation algorithm. It is computationally efficient, requiring approximately five multiplications per sample point. The interpolation concept is extended to the principal component technique for separation of action potential waveforms. The energy function is interpolated and used to align the waveforms, after which the interpolated coefficients can be used for high speed classification. The technique shows an improvement in both alignment error and effective signal/noise ratio in comparison with sampling or interpolation to a voltage peak.     

Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias

We present a detailed statistical analysis of fluorescence correlation spectroscopy for a wide range of timescales. The derivation is completely analytical and can provide an excellent tool for planning and analysis of FCS experiments. The dependence of the signal-to-noise ratio on different measurement conditions is extensively studied. We find that in addition to the shot noise and the noise associated with correlated molecular dynamics there is another source of noise that appears at very large lag times. We call this the "particle noise," as its behavior is governed by the number of particles that have entered and left the laser beam sample volume during large dwell times. The standard deviations of all the points on the correlation function are calculated analytically and shown to be in good agreement with experiments. We have also investigated the bias associated with experimental correlation function measurements. A "phase diagram" for FCS experiments is constructed that demonstrates the significance of the bias for any given experiment. We demonstrate that the value of the bias can be calculated and added back as a first-order correction to the experimental correlation function.     

Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy

We introduce a new analysis technique for fluorescence fluctuation data. Time-integrated fluorescence cumulant analysis (TIFCA) extracts information from the cumulants of the integrated fluorescence intensity. TIFCA builds on our earlier FCA theory, but in contrast to FCA or photon counting histogram (PCH) analysis is valid for arbitrary sampling times. The motivation for long sampling times lies in the improvement of the signal/noise ratio of the data. Because FCA and PCH theory are not valid in this regime, we first derive a theoretical model of cumulant functions for arbitrary sampling times. TIFCA is the first exact theory that describes the effects of sampling time on fluorescence fluctuation experiments. We calculate factorial cumulants of the photon counts for various sampling times by rebinning of the original data. Fits of the data to models determine the brightness, the occupation number, and the diffusion time of each species. To provide the tools for a rigorous error analysis of TIFCA, expressions for the variance of cumulants are developed and tested. We demonstrate that over a limited range rebinning reduces the relative error of higher order cumulants, and therefore improves the signal/noise ratio. The first four cumulant functions are explicitly calculated and are applied to simple dye systems to test the validity of TIFCA and demonstrate its ability to resolve species.     

Mean and variance of ratio estimators used in fluorescence ratio imaging

BACKGROUND: The ratio of two measured fluorescence signals (called x and y) is used in different applications in fluorescence microscopy. Multiple instances of both signals can be combined in different ways to construct different ratio estimators. METHODS: The mean and variance of three estimators for the ratio between two random variables, x and y, are discussed. Given n samples of x and y, we can intuitively construct two different estimators: the mean of the ratio of each x and y and the ratio between the mean of x and the mean of y. The former is biased and the latter is only asymptotically unbiased. Using the statistical characteristics of this estimator, a third, unbiased estimator can be constructed. RESULTS: We tested the three estimators on simulated data, real-world fluorescence test images, and comparative genome hybridization (CGH) data. The results on the simulated and real-world test images confirm the presented theory. The CGH experiments show that our new estimator performs better than the existing estimators. CONCLUSIONS: We have derived an unbiased ratio estimator that outperforms intuitive ratio estimators.     

Increasing turbidity significantly alters the diet of brown trout: a multi‐year longitudinal study

The stomach contents of adult brown trout Salmo trutta from Lake Sorell, Tasmania, were analysed during 6 years of increasing turbidity to follow changes in the diet associated with dramatic changes in water clarity. Brown trout were sampled from 1996, when turbidity was 26 NTU, to 2001 when turbidity was 141 NTU. The mean relative volume of stomach contents decreased progressively to 2001, by which time it was only one sixth of that in 1996, and the mean diversity of prey in stomachs decreased from an average of more than six species per stomach in 1996 to one species in 2001. The species composition of stomach contents shifted from domination by the phreatoicid isopod Colubotelson sp., to the galaxiid fish Galaxias auratus and the amphipod Austrochiltonia australis , and then the cladoceran Daphnia carinata . To give an indication of diet changes over a typical yearly cycle in the current turbid state of the lake, a sample was taken from each season from December 2000 to September 2001. Two basic diets were found during the year; brown trout specialized on D. carinata in summer and autumn, and G. auratus in winter and spring. Mean diversity of prey was less than two species per stomach in all samples from 2000 to 2001, except for the sample from spring 2001 when it was 2·2 species per stomach, and the mean relative volume of stomach contents was more than three times greater in winter than any other season. The ways in which high turbidity may have influenced the changes in the brown trout diet observed since 1996 and the patterns evident during the seasons of 2000–2001 are discussed.     

Signal to noise analysis of multiple color fluorescence imaging microscopy

BACKGROUND: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FISH). With the growing interest in this field, it is important to develop tools for optimizing and estimating the accuracy of different experimental methods. METHODS: We have analyzed the principles of multiple color fluorescence imaging microscopy. First, formalism based on the physical principles of fluorescence microscopy and noise analysis is introduced. Next, a signal to noise (S/N) analysis is performed and summarized in a simple accuracy criterion. The analysis assumes shot noise to be the dominant source of noise. RESULTS: The accuracy criterion was used to calculate the S/N of multicolor FISH (M-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on various types of samples and shows accurate classifications. We have also tested classification accuracy as a function of total measurement time. CONCLUSIONS: The accuracy criterion that we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements is supported by our measurements.     

Optimum signal in a simple neuronal model with signal-dependent noise

How does the information about a signal in neural threshold crossings depend on the noise acting upon it? Two models are explored, a binary McCulloch and Pitts (threshold exceedance) model and a model of waiting time to exceedance--a discrete-time version of interspike intervals. If noise grows linearly with the signal, we find the best identification of the signal in terms of the Fisher information is for signals that do not reach the threshold in the absence of noise. Identification attains the same precision under weak and strong signals, but the coding range decreases at both extremes of signal level. We compare the results obtained for Fisher information with those using related first and second moment measures. The maximum obtainable information is plotted as a function of the ratio of noise to signal.     

Reproductive characteristics ofPseudoperichaeta nigrolineata (Dipt., tachinidae), parasitoid ofOstrinia nubilalis (Lep., pyralidae)

The ovolarviparous tachinid flyPseudoperichaeta nigrolineata Walker is a larval parasitoid of the European corn borer,Ostrinia nubilalis Hübner. A study of the behaviours of gravid females and the kairomones that influence these behaviours led us to quantify several biological characteristics of these females. The females were observed from the moment they became gravid. They were placed in the presence of a corn-caterpillar complex that was changed three times per 24-hour period. Parasitoids lived an average of 8.5 more days. From emergence, the mean longevity was 24 days and the maximum, 39 days. The ovolarviposition period of the sample lasted a total of 12 days. However, the actual number of days of ovolarviposition per female averaged 6.9. The number of days between the end of ovolarviposition and death varies, but 69% of females laid eggs until their death. All the females ovolarviposited; some laid eggs regularly (59%) and others with interruptions (41%). Total mean ovolarviposition per female was 83 maggots, with extremes of 15 and 231 maggots. Thirty percent of the females laid eggs between 30 and 60 planidia. Variability was high for all characteristics studied. Results obtained are discussed with reference to other tachinids and the experimental conditions used.     

Position-sensitive scanning fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.     

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.     

Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism‐related micro‐organisms

Aims: To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism. Methods and Results: Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax (Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10‐μl sample volume onto the sample support was 10⁶ particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 μl, the detection limit could be reduced to 10⁵ particles per ml for spores and to 5 × 10⁴ particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated. Conclusions: The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support. Significance and Impact of the Study: Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique.