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PubMed1999

The photon counting histogram in fluorescence fluctuation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given by Mandel's formula. Based on this equation and considering the fluorescence intensity distribution in the two-photon excitation volume, a theoretical expression for the PCH as a function of the number of molecules in the excitation volume is derived. For a single molecular species two parameters are sufficient to characterize the histogram completely, namely the average number of molecules within the observation volume and the detected photon counts per molecule per sampling time epsilon. The PCH for multiple molecular species, on the other hand, is generated by successively convoluting the photon counting distribution of each species with the others. The influence of the excitation profile upon the photon counting statistics for two relevant point spread functions (PSFs), the three-dimensional Gaussian PSF conventionally employed in confocal detection and the square of the Gaussian-Lorentzian PSF for two photon excitation, is explicitly treated. Measured photon counting distributions obtained with a two-photon excitation source agree, within experimental error with the theoretical PCHs calculated for the square of a Gaussian-Lorentzian beam profile. We demonstrate and discuss the influence of the average number of particles within the observation volume and the detected photon counts per molecule per sampling interval upon the super-Poissonian character of the photon counting distribution.

Diffusion↗
PubMed1999

Analysis of vertical fluorescence resonance energy transfer from the surface of a small-diameter sphere.

Fluorescence resonance energy transfer (FRET) measurements have been used to analyze fluorophore separations in a number of varying geometries, including small particles and extended surfaces. This study focuses on the geometry created by a donor extended above the surface of a small sphere (radius < R0), where the acceptors are integrated into the sphere surface. The model of this geometry was based on an amphipathic molecule with its lipophilic region integrated into a detergent micelle and its hydrophilic region extending outward from the micelle surface, where the donor fluorophore is attached to the hydrophilic region of the molecule. Based on random acceptor incorporation into the micelle, a Poisson distribution was used to calculate the distribution of acceptor probes across the micelle population. The model converges to RET on a flat surface when the radius of the micelle exceeds 0.8 R0. The model was also used to simulate FRET data showing that the positions of donors above the micelle surface could be uniquely resolved. Experimental verification of the model was achieved in a sulfobetaine palmitate micelle with fluorescein isothiocyanate donors attached to detergent-solubilized lipopolysaccharide (LPS) and lipophilic Fast-DiI acceptors. The use of steady-state analysis allowed resolution of cases in which donors were located at different distances from the surface. Combining steady-state with excited-state lifetime analysis allowed resolution of cases where there was a combination of distances. Given the large number of biomolecules that interact with lipids, this approach may prove generally useful for defining molecular conformation.

Biophysical Phenomena↗
PubMed2002

Detection of single molecules: solution-phase single-molecule fluorescence correlation spectroscopy as an ultrasensitive, rapid and reliable system for immunological investigation.

More sensitive techniques in molecular and clinical immunology are essential for the development of reproducible profiles. We have developed a novel methodology named solution-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) that fulfils these demands. It is based on the quantification of the probability density of single molecule events in solution is necessary. For example, the Brownian motion of the fluorophore rhodamine-green is detected. Counting about 100,000 photon counts per second and per molecule permits the identification of one single compound. In order to study the applicability of SPSM-FCS in immunology, we have detected and identified a larger nonfluorescent substance in a very complex mixture. The 'unknown' molecules studied were the autoantibodies in serum samples directed against the antigen alpha 3 chain of type IV collagen. In both systems, we were able to characterize the probability density of single fluorescent molecules by means of the averaged absolute molecule number without any calibration. The specific molecules exhibited a Poisson distribution in solution in terms of their 'critical' bulk concentration below about 1 nM. This proof of principle indicates how the SPSM-FCS methodology could be used in immunoassays.

Animals↗
PubMed1992

The development of a technique to estimate the number of motor units in the urethral sphincter.

An electromyographic technique is presented that has been developed in an effort to quantitate the number of motor units in the urinary sphincter. Sphincter contraction was provoked by electrical stimulation and recorded with a catheter mounted electrode. Incremental stimulation produced corresponding increments of sphincter contraction that were recorded as the magnitude of the sphincter electromyogram. Estimates of the number of motor units in the sphincter were made by analysis of variance of a Poisson distribution. Using this method, the mean number of motor units in the sphincter of five dogs was estimated to be 129 with values ranging from 80 to 182. Anatomical correlation was sought by infiltrating this sphincter with horseradish peroxidase, which is taken up by axonal transport to stain the motor neurons in the spinal cord. The mean number of motor neurons was 229 with values ranging from 116 to 358. There are technical and physiologic explanations for the observed differences between the predicted and the actual number of motor neurons stained and this is addressed in the discussion.

Animals↗
PubMedDate not supplied

The effective number of a population that varies cyclically in size. II. Overlapping generations.

We consider haploid and dioecious age-structured populations that vary over time in cycles of length k. Results are obtained for both autosomal and sex-linked loci if the population is dioecious. It is assumed that k is small in comparison with numbers of haploid individuals (or of numbers of males and females) in any generation of a cycle. The inbreeding effective population size N(e) is then approximately given by the expression [T summation operator (k-1)(j=0)1/[N(e)(j)T(j)]](-1), where N(e)(j) and T(j) are, respectively, the effective population size and generation interval that would hold if the population was at all times generated in the same way as at time j. The constant T, which is the effective overall generation interval, is defined to be k times the harmonic mean of the quantities T(j). Our expressions for T and N(e), in terms of N(e)(j) and T(j), are general, but the N(e)(j)s are derived under the assumption that offspring are produced according to Poisson distributions.

Alleles↗
PubMed2001

[Trends in cancer mortality in Catalonia (1975-1998)].

BACKGROUND: Aimed at assessing recent trends of cancer mortality in Catalonia (Spain), we analysed mortality rates for main tumour locations in men and women between 1975 and 1998. MATERIAL AND METHOD: Mortality data were obtained from the Mortality Registry of Catalonia. For each sex, age-standardized (standard world population) mortality rates were determined by means of the direct method, and generalized lineal models with Poisson distribution were adjusted to obtain annual change percentages. RESULTS: In men, the overall cancer mortality (except skin non-melanoma) was highest within 1991-1992 (age-standardized rate: 181.39/100,000) and decreased thereafter until 169.47/100,000 in 1995-1996. Lung cancer was the main cause of cancer mortality, displaying a 4% annual increase until 1989-1990, and a subsequent stabilization (-0.5% annual between 1989 -1990 and 1997 - 1998); Colorectal cancer was the second cause of cancer death, both in men and women, with a mortality rate stabilization since the 1990s. Prostate cancer became the third cause of cancer death in 1997-1998. In women, overall cancer (except skin non-melanoma) mortality rate was of 77.98/100,000 in 1997-98, showing a 0.9% annual decrease since 1989-1990. Breast cancer was the chief cause of cancer death within 1997-1998: after a sustained increase since 1975-1976, mortality started to decline (-2.0% annual) since 1989-1990. Lung cancer became the third cause of cancer death among women within 1997-1998, just after breast cancer and colorectal cancer, while gastric and uterine cancer became the fourth and fifth cancer death causes during this period, respectively. CONCLUSION: After years of a steady increase, mortality rates for main cancer types stabilized since 1989-1990 in Catalonia.

Adolescent↗
PubMed2003

[Short-term relationship between air pollution and mortality in 13 Spanish cities].

BACKGROUND AND OBJECTIVE: EMECAM is a collaborative project that seeks to evaluate the short-term effects of air pollution on mortality in Spain. MATERIAL AND METHOD: We collected data for air pollutants (particles and gases), daily mortality (total except external, cardiovascular and respiratory causes) and co-variables (temperature, humidity, influenza and calendar variables) in 13 Spanish cities. The magnitude of the association in every city was estimated using GAM under a Poisson distribution. Combined estimates for each cause and pollutant were obtained under 'fixed effects' and 'random effects'models. RESULTS: An increase of 10 (g/m3 in the levels of the average of the concurrent and one day lag for black smoke was associated with a 0.8% (CI: 0.4-1.1) increase in mortality. The same increase in the concentration of SO2 was associated with a 0.5% (CI: 0.1-1.0) increase in daily deaths, and a 0.6% (CI: 0.3-0.8) increase in the case of NO2. An increase of 1 mg/m3 in the levels of CO was associated with an increase of 1.5% (CI: 0.5-2.6) in daily deaths. CONCLUSIONS: There is a short-term association between increases of daily levels of air pollutants and the number of daily deaths in Spanish cities.

Air Pollution↗
PubMed2001

Study of adaptive mutations in Salmonella typhimurium by using a super-repressing mutant of a trans regulatory gene purR.

Salmonella typhimurium purR encodes a transcriptional repressor regulating gene expression of de novo purine nucleotide biosynthesis. It represses purD gene transcription by binding to the 16-base pair purD operator (PUR box). A S. typhimurium strain carrying a super-repressing mutant of purR, purR(s), has been used as an experimental system to study adaptive mutation. Escherichia coli lac genes were genetically engineered into S. typhimurium chromosome and repressed by purR(s) so that they could be used as an indicator of adaptive mutations in purR(s) or in the purD operator. Mutations in purR(s) or in the purD operator accumulated when the mutant strain was placed on a minimal lactose plate supplemented with 10 microg/ml of adenine during prolonged incubation. These specific mutations reverted the mutant strain from lac(-) to lac(+) phenotype. The lac(+) strains were categorized into the early- and late-arising mutants according to the time for colony appearance. Our genetic studies indicate that (i) Poisson distributed mutations accumulated in the chromosomal regulatory gene purR or the purD operator in very slowly dividing cells under selection; (ii) after about 8 days of selection, the frequency of mutations in purD operator reached the high value of about two mutations per 10(8) cells; (iii) the mutational spectrum in the purD operator during growth was not significantly different from that during selection; (iv) defects in mutL or mutS appeared to have a stronger effect on growth-dependent mutations than on adaptive mutations.

Adaptation, Physiological↗
PubMed1999

Estimation of dose in cancer patients treated with fractionated radiotherapy using translocation, dicentrics and micronuclei frequency in peripheral blood lymphocytes.

The frequency of translocation, dicentrics (DC) and micronuclei (MN) was studied in blood samples exposed in vitro to (60)Co gamma radiation and cervical cancer patients undergoing fractionated radiotherapy. The samples exposed under in vitro condition showed that the frequency of translocation and dicentric followed Poisson distribution ('u' varied between -0.04 and +1.41 for translocation and between -0.09 and +1.81 for DC) and that obtained with MN follow over dispersion ('u' varied between +2.04 and +9.28). However, the cervical cancer patients undergoing radiotherapy showed over dispersion for all these three aberrations (DC, MN and translocation). The frequencies of aberrations obtained in cancer patients were found to be lower than those obtained for in vitro exposure for doses more than 2 Gy equivalent whole body dose (EWBD). The dose-response curves were constructed using the frequencies of DC, MN and translocation as a function of EWBD. Doses as measured from the dose response curves were compared with the estimated dose in order to check whether the measured and estimated doses agree. The percent variation between the doses measured from aberration frequencies and that of the estimated dose was lower with translocation (10.8+/-7.41%) compared to those obtained with DC (38. 08+/-31.85%) and MN (47.19+/-31.80%).

Adolescent↗
PubMed2002

The effect of drinking water treatment on the spatial heterogeneity of micro-organisms: implications for assessment of treatment efficiency and health risk.

The effect of drinking water treatment (ferric coagulation, floc blanket clarification, rapid sand filtration) on the spatial heterogeneity of five species of micro-organism was studied at pilot scale. It was found that the spatial heterogeneity of vegetative bacteria (namely total coliform and heterotrophic (22 degrees C; 3 d) bacteria) was little affected by treatment. Indeed, counts of total coliform bacteria within 500 l volumes of treated water were Poisson distributed (i.e. showed minimum variation). In contrast, treatment appeared to increase the spatial heterogeneity (or clustering) of both aerobic spores indigenous to the raw water and Bacillus subtilis var niger spores added to the raw water. Furthermore, B. subtilis var niger spores added to the raw water were detected in the treated water 25 h after termination of spiking to the raw water. The effect on C. parvum oocysts added to the raw water could not be determined because few oocysts broke through treatment into the treated water. Indeed oocyst removals of 5-6 logs were apparent. "Species-specific" differences in the removal ratios were also demonstrated. It is concluded that audits for treatment processes based on single 100 ml "spot" samples for spores will tend to over-estimate the net spore removal and hence underestimate the public health risk. Spatial heterogeneity of counts in treated water contributes to explaining why no "ideal" surrogate has been identified for treatment plant performance.

Animals↗
PubMed1998

Down's syndrome, ageing and fragile sites.

Fragile sites have been interesting for mapping chromosomal regions involved in disease and ageing. The chromosomal fragile site expression from 38 Down's syndrome (DS) individuals aged 0-48 years was investigated in blood peripheral lymphocytes. Fragile sites were statistically characterized as the minimum expected number of lesions per band based on a Poisson distribution. The results showed that the fragile site 2q11 was associated with the DS condition and fragile sites 5q31, 6p21 and 9q12 with ageing in DS subjects. Fragility in 6p21 has also been associated with Alzheimer's disease patients.

Adolescent↗
PubMed2003

Second primary cancers after cervical intraepithelial neoplasia III and invasive cervical cancer in Southeast England.

OBJECTIVE: Multiple primary cancers may arise in an individual because they share a common environmental risk factor (such as smoking); genetic predisposition or immunodeficiency may predispose to both cancers, or treatment for one cancer may cause a second cancer. The objective of this analysis was to identify which, if any, cancers occur more often than would be expected in a cohort of women diagnosed with cervical intraepithelial neoplasia III (CIN III) and in women with invasive cervical cancer. METHODS: The Thames Cancer Registry was used to identify two cohorts of women diagnosed with either CIN III or invasive cervical cancer. The number of subsequent cancers at other sites was observed and compared to the expected number, based on relevant age-, sex-, and period-specific incidence rates. Standardised incidence rates (SIRs) were calculated assuming a Poisson distribution. RESULTS: The following cancer sites were significantly increased after a diagnosis of either CIN III or cervical cancer: anus (SIR 5.9 and 6.3, respectively), lung (SIR 1.8 and 2.5), vulva (SIR 4.4 and 1.9), vagina (SIR 18.5 and 8.0), and kidney (SIR 1.6 and 1.9). In addition, the incidence of cancers of the rectum, bladder, and connective tissue was significantly increased after invasive cervical cancer. Cervical cancers were seen significantly more often than expected after cancers of the anus and vagina. CONCLUSION: These results support the hypothesis that cancers of the cervix, anus, vulva, and vagina share common risk factors such as human papillomavirus and smoking.

Adult↗
PubMed2002

Simulation model of within-herd transmission of bovine tuberculosis in Argentine dairy herds.

Transmission of bovine tuberculosis was quantified in three dairy herds located in south Santa Fe Province, Argentina. Using estimates of Mycobacterium bovis transmission (beta) and a Reed-Frost simulation model, the prevalence of tuberculosis infection in the study herds over time was investigated. The Reed-Frost model was modified by incorporating randomness in both beta and the incubation period (alpha) of M. bovis. The mean estimated herd beta was 2.2 infective contacts per year and did not differ significantly between the study herds. Modeling alpha as Poisson distributed (mean 24 months) best fit the observed prevalences. Infection was predicted by the model either to spread quickly (<10 years) within a herd and reach a high prevalence (>50%), or to persist at a low prevalence (<5-10%). The model was robust, predictions were realistic and the mean beta estimated was consistent with previous studies of bovine tuberculosis.

Animal Husbandry↗
PubMed2002

Focal stimulation of single GABAergic presynaptic boutons on the rat hippocampal neuron.

Evoked inhibitory postsynaptic currents (eIPSCs) generated from a single GABAergic bouton were recorded and the functional properties were investigated. Native single boutons attached to mechanically dissociated rat hippocampal CA1 neurons, namely "synaptic bouton" preparation, were visualized with FM 1-43 dye and selectively stimulated by a glass pipette directed to a single bouton by focal stimulation. The GABAergic eIPSCs were elicited in like all-or-none fashion regarding both stimulus strength and pipette location, thus indicating that the eIPSCs result from the activation of a single bouton. The GABA release from the boutons was action potential dependent since eIPSCs were blocked in the presence of either voltage-dependent Na(+) or Ca(2+)channel blocker. Even in the presence of tetrodotoxin (TTX), eIPSCs could be elicited by additional application of a voltage-dependent K(+) channel blocker, 4-AP. The GABA release depended on external Ca(2+) concentration. Amplitude histogram of eIPSCs did not follow Poisson distribution or show discrete peaks. As a result, this new experimental approach using both focal stimulation and a synaptic bouton preparation allows for a detailed study of the native synaptic machinery in nerve terminals measuring smaller than 1 microm in size in the CNS.

Action Potentials↗
PubMed2002

Probability of cell hits in selected organs and tissues by high-LET particles at the ISS orbit.

The fluence of high-LET particles (HLP) with LET infinity H2O greater than 15 keV micrometers-1 in selected organs and tissues were measured with plastic nuclear track detectors using a life-size human phantom on the 9th Shuttle-Mir Mission (STS-91). The planar-track fluence of HLP during the 9.8-day mission ranged from 1.9 x 10(3) n cm-2 (bladder) to 5.1 x 10(3) n cm-2 (brain) by a factor of 2.7. Based on these data, a probability of HLP hits to a matured cell of each organ or tissue was roughly estimated for a 90-day ISS mission. In the calculation, all cells were assumed to be spheres with a geometric cross-sectional area of 500 micrometers2 and the cell-hit frequency from isotropic space radiation can be described by the Poisson-distribution function. As results, the probability of one or more than 1 hit to a single cell by HLP for 90 days ranged from 17% to 38%; that of two or more than 2 hits was estimated to be 1.3-8.2%.

Brain↗
PubMedDate not supplied

Sonicated echocardiographic contrast agents: reproducibility studies.

This article describes the production, analysis, and reproducibility of forming microbubbles for contrast ultrasound imaging. The sonication method used to generate microbubbles was tested by four independent observers, and a subsequent laser particle counter analysis of microbubble size and concentration determined the reproducibility of the method. The results indicated that the mean bubble size was 3.3 +/- 1.2 microns for the entire group, based on three trials of each of the four participants. The characteristics of the bubble size of the microbubbles between observers were assessed with a Poisson distribution with the reproducibility based on the sample mean for each observer's trials. Standardization and calibration of the laser particle counter was accomplished with commercially available latex spheres, sonicated albumin microspheres, and a Coulter counter analysis. Our results indicate that the sonication technique generates small microbubbles with a reproducible uniform size distribution. The method of microbubble production is reproducible and can be widely applied for use in contrast echocardiographic perfusion imaging of tissue in a variety of research and clinical studies.

Air↗
PubMed2000

A study of the effect of measurement error in predictor variables in nondestructive assay

It is not widely known that ordinary least squares estimates exhibit bias if there are errors in the predictor variables. For example, enrichment measurements are often fit to two predictors: Poisson-distributed count rates in the region of interest and in the background. Both count rates have at least random variation due to counting statistics. Therefore, the parameter estimates will be biased. In this case, the effect of bias is a minor issue because there is almost no interest in the parameters themselves. Instead, the parameters will be used to convert count rates into estimated enrichment. In other cases, this bias source is potentially more important. For example, in tomographic gamma scanning, there is an emission stage which depends on predictors (the "system matrix") that are estimated with error during the transmission stage. In this paper, we (1) provide background information for the impact and treatment of errors in predictors, (2) present results of candidate methods of compensating for the effect, (3) review some of the nondestructive assay situations where errors in predictors occurs, and (4) provide guidance for when errors in predictors should be considered in nondestructive assay.

Journal Article↗