Quantitative and qualitative profiling of mitochondrial DNA length heteroplasmy

Quantitative and qualitative analysis of mitochondrial DNA length heteroplasmy for the first hypervariable segment (HV1) and second hypervariable segment (HV2) regions were performed using size-based separation of fluorescently-labeled polymerase chain reaction (PCR) products by capillary electrophoresis. In this report, the relative proportions of length heteroplasmies in individuals were determined, and each length variant in the heteroplasmic mtDNA mixture was identified. The study demonstrated that 36% and 69% of Koreans show length heteroplasmy in the HV1 and HV2 regions, respectively. Electropherograms revealed that length heteroplasmy in the HV1 region resulted in over 5 length variants in an individual. The peak patterns of length heteroplasmy in the HV1 region were classified into five major types. In the HV2 region, length heteroplasmy resulted in 3-6 length variants in an individual, and showed seven variant peak patterns. The increased knowledge concerning mtDNA length heteroplasmy is believed to not only offer a useful means of determining genetic identity due to increased mitochondrial DNA haplotype diversity by allowing mtDNAs to be classified into several peak patterns, but also represent a promising tool for the diagnosis of several common diseases which are etiologically or prognostically associated with mtDNA polymorphisms.     

Extremely high levels of human mitochondrial DNA heteroplasmy in single hair roots

For many years it has been assumed that the vast majority of mitochondrial genomes of a single individual are identical, both in the same tissue and within different tissues. Incidences of heteroplasmy (i.e., the occurrence of two or more codominating types of molecules within the mitochondrial DNA population of the same individual) were thought to be extremely rare. This study strongly supports the thesis that heteroplasmy is a principle, rather than an exception, in mitochondrial DNA genetics. During direct sequencing of the first hypervariable segment of the human mitochondrial control region (HV1) in 100 single hair roots obtained from 35 individuals, 24 different heteroplasmic positions were identified. Unusually high levels of heteroplasmy (up to six positions in the HV1 region) were encountered in two individuals. Two individuals related in maternal lineage shared the same heteroplasmic positions. Moreover, highly variable levels of heteroplasmy were observed even among roots from the same individual. The most probable mechanisms involved in generating so many mismatches are mutations occurring presumably in the female germline, followed by differential segregation of mitotypes during the development of individual hairs. Generally, heteroplasmy complicates sequence comparisons in mitochondrial DNA testing performed for forensic purposes, but in some cases it can substantially increase the discriminating power of the analysis.     

High levels of mitochondrial DNA heteroplasmy in single hair roots: reanalysis and revision.

The present study demonstrates a reinvestigation of the mitochondrial DNA sequence heteroplasmy, which was previously found by the use of nested polymerase chain reaction (PCR) technique in single hairs of 13 individuals. The direct PCR approach was used for the amplification of mitochondrial DNA and a phylogenetic analysis was applied to both data sets for the verification of the authenticity of sequences. The comparative analysis of the sequencing results obtained from the same hair DNA extracts - but using two different techniques - shows that direct mitochondrial DNA amplification results in a considerably lower number of mixed positions. The majority of the confirmed heteroplasmic variants preferentially occurs in mitochondrial DNA hypervariable sites (mutational hotspots). However, the pattern of heteroplasmic mutations observed in four extracts after nested PCR significantly differs from the pattern of natural mutations. Some of these rare polymorphisms should be revised as inconsistent with phylogenetic expectations. The results of the present study contribute to the earlier reports by indicating that phylogenetic analysis is an effective tool in a posteriori quality check of mitochondrial DNA data.     

Applications of micro-analysis to individual environmental particles

An overview is given of the recent applications of micro-analytical techniques to single particle environmental research performed at the University of Antwerp since 1990. Automated electron probe X-ray micro-analysis, laser microprobe mass spectrometry and micro-particle induced X-ray emission are the techniques most used for aerosol, aqueous suspension and sediment characterisation. Other techniques like scanning transmission electron microscopy, electron energy loss spectroscopy, Fourier transform infra red microscopy and secondary ion mass spectrometry have only recently been implemented into environmental research.     

Applications of micro-analysis to individual environmental particles

An overview is given of the recent applications of micro-analytical techniques to single particle environmental research performed at the University of Antwerp since 1990. Automated electron probe X-ray micro-analysis, laser microprobe mass spectrometry and micro-particle induced X-ray emission are the techniques most used for aerosol, aqueous suspension and sediment characterisation. Other techniques like scanning transmission electron microscopy, electron energy loss spectroscopy, Fourier transform infra red microscopy and secondary ion mass spectrometry have only recently been implemented into environmental research.     

Micro-FTIR measurements on individual interplanetary dust particles

The unique characteristics of micro-FTIR spectroscopy for the study of individual interplanetary dust particles (IDPs) are discussed: (1) quick and nondestructive identification of dominant silicate phases (pyroxenes, olivines, layer lattice silicates) and selection of unique IDPs for subsequent intensive study by complementary analytical techniques; (2) identification of potential carbonaceous carrier phases of isotopic anomalies; (3) measurement of IR absorption features in IDPs that can be compared with IR emission features obtained by telescopic observations from comets and protostars.     

Determination of the effective charge of individual colloidal particles

An optical method is presented that allows simultaneous determination of the diffusion constant and electrophoretic mobility of individual charged particles with radius down to 0.2 mum. By this method the size dependency of the effective charges and zeta potentials of individual particles can be investigated, as well as interparticle interactions and Brownian motion in confined geometries. The diffusion constant and mobility are determined from the power spectrum of the particle speed in a sinusoidal electrical field. The accuracy of the method was tested on PMMA spheres of known size in water. Experiments have been carried out on charged pigment particles with low concentration in a nonaqueous medium containing a charging agent. The mobility is found to be independent of the particle size.     

Altering surface charge nonuniformity on individual colloidal particles

Charge nonuniformity (sigmazeta) was altered on individual polystyrene latex particles and measured using the novel experimental technique of rotational electrophoresis. It has recently been shown that unaltered sulfated latices often have significant charge nonuniformity (sigmazeta = 100 mV) on individual particles. Here it is shown that anionic polyelectrolytes and surfactants reduce the native charge nonuniformity on negatively charged particles by 80% (sigmazeta = 20 mV), even while leaving the average surface charge density almost unchanged. Reduction of charge uniformity occurs as large domains of nonuniformity are minimized, giving a more random distribution of charge on individual particle surfaces. Targeted reduction of charge nonuniformity opens new opportunities for the dispersion of nanoparticles and the oriented assembly of particles.     

Spreading of individual toner particles studied using in situ optical microscopy

This study develops and tests an experimental method to monitor in situ the dynamic spreading of individual toner particles on model substrates during heating, to simulate on laboratory scale the fusing sub-processes occurring in electrophotographic printing of paper. Real toner particles of cyan, magenta, yellow and black are transformed to perfect spheres by a temperature pre-treatment, then applied to the substrate, either high-energy clean glass or low-energy hydrophobised glass, and heated at rates up to 50 degrees C/min. The subsequent spreading as a function of time (and temperature) is recorded by an optical microscope and CCD camera mounted above the substrate, with the measured drop covering area used to calculate the corresponding toner-substrate-air contact angle. On the hydrophobic substrate the spreading is limited and equal for all four colours, while the substantially greater spreading on the hydrophilic substrate is accompanied by significant differences between the toner colours. In particular, the cyan and black toners are found to spread to almost twice the extent of the yellow particles. The dynamic spreading behaviour is interpreted in terms of complementary measurements of substrate and toner surface energy components and bulk toner rheology, and a simple empirical relation is proposed that fits very well the measurements for all toner and substrate types tested. In particular, the spreading relation is found to be determined only by the toner surface energy and its equilibrium contact angle, with no explicit dependence on toner viscosity.     

Sampling and analysis of individual particles by aerosol mass spectrometry

Over the past decade, aerosol mass spectrometry has developed into a powerful method for characterizing individual particles in air. Recent advances in the design of inlets and mass spectrometers have extended the size range of particles that can be analyzed. In this tutorial, fundamental aspects of particle motion in sampling inlets are introduced. Basic experimental configurations for achieving a high analysis rate and the ability of laser ablation to provide chemical composition information are reviewed. An example of the use of this technology to study atmospheric phenomena is also presented. Significant opportunity exists for designing new experiments at the interface of aerosol mass spectrometry and conventional molecular mass spectrometry.     

Nitrogen-skinned carbon nanocone enables non-dynamic electrochemistry of individual metal particles

Science China Chemistry - Nano-impact electrochemistry is an efficient way to probe the physical and chemical properties of individual particles. Unfortunately, limited by the weak adsorption...     

Characterization of individual atmospheric particles by element mapping in electron probe microanalysis

In this paper procedures for the characterization of individual aerosol particles by element mapping in the electron microprobe are presented. The number, size and qualitative chemical composition of particles is derived from a combination of secondary or backscattered electron images and element distribution maps. Accuracy of the size distribution and reliability of the qualitative analysis procedure were checked with silicate samples. In order to obtain a semi-quantitative estimate of the chemical composition of individual particles the count rates taken from element distribution maps are corrected for matrix and geometric effects using particle ZAF procedures.Dedicated to Professor Dr. rer. nat. Dr. h.c. Hubertus Nickel on the occasion of his 65th birthday     

Source identification of lead-containing particles in the ambient air of the center of Shanghai by analyzing individual aerosol particles

Summary Single aerosol particles were analyzed in the ambient air of the center of Shanghai by scanning proton microprobe to obtain characteristic X-ray spectra (micro-PIXE) which were considered to be the fingerprints of these aerosol particles. The origin of the lead-containing particles was identified by the combination of the micro-PIXE spectra with pattern recognition technique. It was found that the most of the lead-containing particles were derived from vehicle exhaust, coal combustion and soil dust.     

Detection of individual insulating entities by electrochemical blocking

Electrochemical blocking is a type of single-entity electrochemical measurement particularly well adapted to the detection of insulating particles. The digital detection of ultralow concentrations of artificial entities such as polymer particles or biotargets such as proteins and bacteria represents an exceptional opportunity for sensing applications. In this review, we explore the latest development in the field of electrochemical blocking and propose some perspectives.     

Characterization of individual airborne particles - methods used and their limitations

The characterization of individual airborne particles plays an important role in assessing environmental contamination. The morphology, elemental composition and surface properties of these particles must be determined for complete characterization.     

Automated analysis of individual particles using a commercial capillary electrophoresis system

Capillary electrophoretic analysis of individual submicrometer size particles has been previously done using custom-built instruments. Despite that these instruments provide an excellent signal-to-noise ratio for individual particle detection, they are not capable of performing automated analyses of particles. Here we report the use of a commercial Beckman P/ACE MDQ capillary electrophoresis (CE) instrument with on-column laser-induced fluorescence (LIF) detection for the automated analysis of individual particles. The CE instrument was modified with an external I/O board that allowed for faster data acquisition rates (e.g. 100 Hz) than those available with the standard instrument settings (e.g. 4 Hz). A series of eight hydrodynamic injections expected to contain 32 +/- 6 particles, each followed by an electrophoretic separation at -300 V cm(-1) with data acquired at 100 Hz, showed 28 +/- 5 peaks corresponding to 31.9 particles as predicted by the statistical overlap theory. In contrast, a similar series of hydrodynamic injections followed by data acquisition at 4 Hz revealed only 8 +/- 3 peaks suggesting that the modified system is needed for individual particle analysis. Comparison of electropherograms obtained at both data acquisition rates also indicate: (i) similar migration time ranges; (ii) lower variation in the fluorescence intensity of individual peaks for 100 Hz; and (iii) a better signal-to-noise ratio for 4 Hz raw data. S/N improved for 100 Hz when data were smoothed with a binomial filter but did not reach the S/N values previously reported for post-column LIF detection. The proof-of-principle of automated analysis of individual particles using a commercially available CE system described here opens exciting possibilities for those interested in the study and analyses of organelles, liposomes, and nanoparticles.     

Synthesis of monodisperse fluorescent core-shell silica particles using a modified Stober method for imaging individual particles in dense colloidal suspensions

Core-shell silica particles, with a diameter of 1.5 mum, containing a dye fluorescein isothiocyanate (FITC), are synthesized by the hydrolysis and condensation of tetraethylorthosilicate (TEOS). Sodium dodecyl sulfate (SDS) is added to synthesize fluorescent core particles with the diameter of approximately 1 mum. In the addition of SDS, the surface charge reduced by counterions (Na+) of the surfactant leads to a higher degree of aggregation of the primary particles and the formation of larger secondary particles. The particle growth kinetics confirms the aggregation growth model for the synthesis of monodisperse silica particles, and also shows the dependence of final particle size on colloidal stability resulting from the addition of SDS. Light and X-ray scattering data reveal that the final particles have compactly packed structures with smooth surfaces. The seeded growth technique is then used to form a silica shell layer on the fluorescent core. The added amount of water and NH4OH has significant effects on shell formation. Finally, the final core-shell silica particles are modified by chemisorption of octadecanol at the surface to be dispersed in organic solvents. Octadecyl-coated silica particles are sterically stabilized in silica index-matching solvents such as chloroform and hexadecane to directly image separate particles using confocal microscopy. In chloroform, the organophilic silica particles disperse well, whereas in hexadecane they form a volume-filling gel structure at room temperature.     

The present state of local analysis: Analysis of individual small particles

Summary This paper tries to give a survey of the present state of local analysis, using as an example the problem of chemical identification of individual inorganic airborne particles, which can have a widely varying composition and which are always present as an extremely complex mixture. It is shown that by application of the different electron probe techniques evaluating a variety of analytical signals such as inner orbital and valence band X-ray spectra, electron diffraction patterns, electron energy-loss spectra and secondary and transmission electron images the size range of particle identification and characterization can be extended towards diameters as small as 100 Å The analytical possibilities are treated, as well as the limitations associated with each of the techniques.

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Zusammenfassung Es wird versucht, einen Überblick über den gegenwärtigen Stand der Lokalanalyse an Hand der chemischen Identifizierung einzelner anorganischer Luftpartikel zu geben, die sehr unterschiedlich zusammengesetzt sein können und stets in Form sehr komplexer Gemische auftreten. Durch Anwendung der verschiedenen Möglichkeiten der Elektronenstrahlmikroanalyse und Auswertung verschiedener analytischer Signale, wie der Innerorbital- und der Valenzband-Röntgenspektren, der Elektronenbeugungsdiagramme, der Elektronenenergieverlust-Spektren sowie der Bilder der Sekundär- und transmittierten Elektronen, kann die Identifizierung und Charakterisierung bis zu Teilchendurchmessern von 100 Å ausgedehnt werden. Die analytischen Möglichkeiten und die Grenzen der besprochenen Verfahren wurden dargelegt.

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Presented at the International Symposium on Microchemical Techniques, May 23–27, 1977, Davos, Switzerland.     

Problems in quantitatively analyzing individual salt aerosol particles using electron energy loss spectroscopy

Polydisperse standard aerosols of NaCl, (NH₄)₂SO₄ and KNO₃ have been generated from their solutions by pneumatic nebulization. These aerosol particles are electron beam sensitive and therefore special precautions are necessary. A methodology is proposed for serially recording electron energy loss spectra (EELS) from sub-micrometer salt particles. The results of quantification are compared with those obtained by parallel electron energy loss spectroscopy (PEELS).Experimental conditions such as the intensity of the primary beam, time of radiation and temperature are responsible for large deviations from the theoretical values. These deviations arise because heavy mass losses occur, especially during the serial spectrum acquisition. The more time consuming serial EELS is therefore at a disadvantage relative to the parallel method.The best results are obtained for NaCl because the halogen loss can be reduced more efficiently than nitrogen and oxygen losses. The results show that nitrogen loss occurs within the first few seconds of the experiment at normal radiation doses. Even at cryogenic temperatures, losses of volatile elements cannot be avoided.     

Comparison of ICP-MS and SIMS techniques for determining uranium isotope ratios in individual particles

The determination of uranium isotope ratios in individual particles is of great importance for nuclear safeguards. In the present study, an analytical technique by inductively coupled plasma mass spectrometry (ICP-MS) with a desolvation sample introduction system was applied to isotope ratio analysis of individual uranium particles. In ICP-MS analysis of individual uranium particles with diameters ranging from 0.6 to 4.2 μm in a standard reference material (NBL CRM U050), the use of the desolvation system for sample introduction improved the precision of ²³⁴U/²³⁸U and ²³⁶U/²³⁸U isotope ratios. The performance of ICP-MS with desolvation was compared with that of a conventionally used method, i.e., secondary ion mass spectrometry (SIMS). The analysis of test swipe samples taken at nuclear facilities implied that the performance of ICP-MS with desolvation was superior to that of SIMS in a viewpoint of accuracy, because the problems of agglomeration of uranium particles and molecular ion interferences by other elements could be avoided. These results indicated that ICP-MS with desolvation has an enough ability to become an effective tool for nuclear safeguards.