Super-resolution imaging and estimation of protein copy numbers at single synapses with DNA-PAINT

In the brain, the strength of each individual synapse is defined by the complement of proteins present or the "local proteome." Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments

Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion

Single-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex-consisting of talin, kindlin and active beta 1-integrin-that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells. Single-molecule localisation microscopy is limited by low labeling and detection efficiencies of the molecular probes. Here the authors report a framework to obtain absolute molecular quantities on a true molecular scale; the data reveal a ternary adhesion complex underlying cell-matrix adhesion

Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes

Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis

A biotecnologia nos programas de melhoramento de forrageiras tropicais da Embrapa Gado de Corte.

Neste trabalho procurou-se apresentar e discutir, de forma ampla, o uso da biotecnologia e seu potencial para os programas de melhoramento de forrageiras tropicais realizados na Embrapa Gado de Corte. O uso da biotecnologia nesses programas é uma atividade recente, mas importantes resultados vêm sendo gerados a fim de auxiliar o processo de obtenção de novas cultivares forrageiras. A maioria dos trabalhos apresentados utiliza marcadores Random Amplification of Polymorphic DNA (RAPD) para aplicações em curto prazo: estudos de diversidade em acessos de bancos de germoplasma, identificação de híbridos e estimação da taxa de cruzamento. Aplicações em médio e longo prazos do uso de marcadores, como mapeamento genético e seleção auxiliada por marcadores moleculares (SAMM), ainda necessitam de maiores investimentos, tanto na busca de novos marcadores, quanto no desenvolvimento de populações adequadas para esses estudos. Em 2007, teve início uma nova linha de pesquisa nessa unidade, a prospecção de genes com características econômicas. Genes para a tolerância ao alumínio são o foco dessa pesquisa que pretende explorar a sintenia entre os genomas de gramíneas, visando ao desenvolvimento de cultivares de braquiária mais tolerantes ao alumínio. A Embrapa Gado de Corte vem investindo em pessoal e aquisição de equipamentos para avançar não só na produção de cultivares de forrageiras mais adaptadas às necessidades de um mercado cada vez mais exigente, como também no crescimento institucional do setor de biotecnologia.bitstream/CNPGC-2009-09/12403/1/DOC168.pd

Investigations of Ra+^+ properties to test possibilities of new optical frequency standards

The present work tests the suitability of the narrow transitions $7s \ ^2S_{1/2} \to 6d ^2D_{3/2}$and$7s ^2S_{1/2} \to 6d ^2D_{5/2}$in Ra$^+$for optical frequency standard studies. Our calculations of the lifetimes of the metastable$6d$states using the relativistic coupled-cluster theory suggest that they are sufficiently long for Ra$^+$ to be considered as a potential candidate for an atomic clock. This is further corroborated by our studies of the hyperfine interactions, dipole and quadrupole polarizabilities and quadrupole moments of the appropriate states of this system.Comment: Latex files, 5 pages, 1 figur

Double- to Single-Strand Transition Induces Forces and Motion in DNA Origami Nanostructures

The design of dynamic, reconfigurable devices is crucial for the bottom-up construction of artificial biological systems. DNA can be used as an engineering material for the de-novo design of such dynamic devices. A self-assembled DNA origami switch is presented that uses the transition from double- to single-stranded DNA and vice versa to create and annihilate an entropic force that drives a reversible conformational change inside the switch. It is distinctively demonstrated that a DNA single-strand that is extended with 0.34 nm per nucleotide - the extension this very strand has in the double-stranded configuration - exerts a contractive force on its ends leading to large-scale motion. The operation of this type of switch is demonstrated via transmission electron microscopy, DNA-PAINT super-resolution microscopy and darkfield microscopy. The work illustrates the intricate and sometimes counter-intuitive forces that act in nanoscale physical systems that operate in fluids

Calculation of energy levels and transition amplitudes for barium and radium

The radium atom is a promising system for studying parity and time invariance violating weak interactions. However, available experimental spectroscopic data for radium is insufficient for designing an optimal experimental setup. We calculate the energy levels and transition amplitudes for radium states of significant interest. Forty states corresponding to all possible configurations consisting of the $7s$, $7p$ and $6d$ single-electron states as well as the states of the $7s8s$, $7s8p$ and $7s7d$ configurations have been calculated. The energies of ten of these states corresponding to the $6d^2$, $7s8s$, $7p^2$, and $6d7p$ configurations are not known from experiment. Calculations for barium are used to control the accuracy.Comment: 12 pages, 4 table

Комплексное моделирование виброакустических, тепловых и динамических возмущений в трубопроводе, подверженном воздействию климатических условий

Electrical borehole wall images represent grey-level-coded micro-resistivity measurements at the borehole wall. Different scientific methods have been implemented to transform image data into quantitative log curves. We introduce a pattern recognition technique applying texture analysis, which uses second-order statistics based on studying the occurrence of pixel pairs. We calculate so-called Haralick texture features such as contrast, energy, entropy and homogeneity. The supervised classification method is used for assigning characteristic texture features to different rock classes and assessing the discriminative power of these image features. We use classifiers obtained from training intervals to characterize the entire image data set recovered in ODP hole 1203A. This yields a synthetic lithology profile based on computed texture data. We show that Haralick features accurately classify 89.9% of the training intervals. We obtained misclassification for vesicular basaltic rocks. Hence, further image analysis tools are used to improve the classification reliability. We decompose the 2D image signal by the application of wavelet transformation in order to enhance image objects horizontally, diagonally and vertically. The resulting filtered images are used for further texture analysis. This combined classification based on Haralick features and wavelet transformation improved our classification up to a level of 98%. The application of wavelet transformation increases the consistency between standard logging profiles and texture-derived lithology. Texture analysis of borehole wall images offers the potential to facilitate objective analysis of multiple boreholes with the same lithology