Beate Sick

Scanning near-field optical microscopy with aperture probes: Fundamentals and applications

In this review we describe fundamentals of scanning near-field optical microscopy with aperture probes. After the discussion of instrumentation and probe fabrication, aspects of light propagation in metal-coated, tapered optical fibers are considered. This includes transmission properties and field distributions in the vicinity of subwavelength apertures. Furthermore, the near-field optical image formation mechanism is analyzed with special emphasis on potential sources of artifacts. To underline the prospects of the technique, selected applications including amplitude and phase contrast imaging, fluorescence imaging, and Raman spectroscopy, as well as near-field optical desorption, are presented. These examples demonstrate that scanning near-field optical microscopy is no longer an exotic method but has matured into a valuable tool.

High-quality near-field optical probes by tube etching

A method called tube etching for the fabrication of near-field optical probes is presented. Tip formation occurs inside a cylindrical cavity formed by the polymer coating of an optical fiber which is not stripped away prior to etching in hydrofluoric acid. The influence of temperature, etchant concentration, and fiber type on the tip quality is studied. A tip formation mechanism for the given geometry is proposed. The procedure overcomes drawbacks of the conventional etching techniques while still producing large cone angles: (i) tips with reproducible shapes are formed in a high yield, (ii) the surface roughness on the taper is drastically reduced, and (iii) the tip quality is insensitive to vibrations and temperature fluctuations during the etching process. After aluminum coating, optical probes with well-defined apertures are obtained. Due to the smooth glass surface the aluminum coating is virtually free of pinholes.

Dysregulation of Gene Expression in Primary Neuron Models of Huntington's Disease Shows That Polyglutamine-Related Effects on the Striatal Transcriptome May Not Be Dependent on Brain Circuitry

Gene expression changes are a hallmark of the neuropathology of Huntingtons disease (HD), but the exact molecular mechanisms of this effect remain uncertain. Here, we report that in vitro models of disease comprised of primary striatal neurons expressing N-terminal fragments of mutant huntingtin (via lentiviral gene delivery) faithfully reproduce the gene expression changes seen in human HD. Neither viral infection nor unrelated (enhanced green fluorescent protein) transgene expression had a major effect on resultant RNA profiles. Expression of a wild-type fragment of huntingtin [htt171-18Q] also caused only a small number of RNA changes. The disease-related signal in htt171-82Q versus htt171-18Q comparisons was far greater, resulting in the differential detection of 20% of all mRNA probe sets. Transcriptomic effects of mutated htt171 are time- and polyglutamine-length dependent and occur in parallel with other manifestations of polyglutamine toxicity over 4–8 weeks. Specific RNA changes in htt171-82Q-expressing striatal cells accurately recapitulated those observed in human HD caudate and included decreases in PENK (proenkephalin), RGS4 (regulator of G-protein signaling 4), dopamine D1 receptor (DRD1), DRD2, CNR1 (cannabinoid CB1 receptor), and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32; also known as PPP1R1B) mRNAs. HD-related transcriptomic changes were also observed in primary neurons expressing a longer fragment of mutant huntingtin (htt853-82Q). The gene expression changes observed in cultured striatal neurons are not secondary to abnormalities of neuronal firing or glutamatergic, dopaminergic, or brain-derived neurotrophic factor signaling, thereby demonstrating that HD-induced dysregulation of the striatal transcriptome might be attributed to intrinsic effects of mutant huntingtin.

RACE: Remote Analysis Computation for gene Expression data.

The Remote Analysis Computation for gene Expression data (RACE) suite is a collection of bioinformatics web tools designed for the analysis of DNA microarray data. RACE performs probe-level data preprocessing, extensive quality checks, data visualization and data normalization for Affymetrix GeneChips. In addition, it offers differential expression analysis on normalized expression levels from any array platform. RACE estimates the false discovery rates of lists of potentially regulated genes and provides a Gene Ontology-term analysis tool for GeneChip data to support the biological interpretation and annotation of results. The analysis is fully automated but can be customized by flexible parameter settings. To offer a convenient starting point for subsequent analyses, and to provide maximum transparency, the R scripts used to generate the results can be downloaded along with the output files. RACE is freely available for use at .

Probing confined fields with single molecules and vice versa.

Single dye molecules are used as local probes to map the spatial distribution of the squared electric field components in the focus of a high numerical aperture lens. Simulated field distributions are quantitatively verified by experimentally obtained fluorescence excitation maps. We show that annular illumination can be used to engineer the field distribution in the focus at a dielectric/air interface such that electric field components in all directions acquire comparable magnitudes. The 3D orientation of molecular absorption dipoles can be determined by comparing measured to simulated image patterns. The presence of longitudinal electric field components in a focus is of particular interest in tip‐enhanced scanning near‐field optical microscopy.

HIGH PHOTO-STABILITY OF SINGLE MOLECULES IN AN ORGANIC CRYSTAL AT ROOM TEMPERATURE OBSERVED BY SCANNING CONFOCAL OPTICAL MICROSCOPY

tum eA ciency lower than 1.2 10 8 . On average, with a detection eA ciency of 6.5%, a stable ¯uorescence signal of 10 5 photons s 1 could be detected fromsingle molecules during 1min of continuous photo-excitation. Both irreversible and reversible abruptuorescence intensity jumps to the background have been observed. The experimental data indicate that diA using quenchers at low concentration in the crystal contribute touorescence bleaching of single molecules. All molecularuorescence signals follow a typical power saturation law with a mean saturation count rate of 4.3 10 5 photons s 1 . The large photo-stability allows for long illumination times and high emission rates of single molecules trapped in a crystal at room temperature. A potential application as single organic quantum light sources under ambient conditions is conceivable.

Three-dimensional optical polarization tomography of single molecules

We apply the concept of tomography to polarization-sensitive optical microscopy of single fluorophores to determine the three-dimensional orientation of molecular absorption dipoles with isotropic sensitivity. Wide-field microscopy provides the opportunity to monitor simultaneously three-dimensional rotation and two-dimensional translation of many molecules in parallel. For orientation determination the molecules are illuminated from different directions of incidence with linearly polarized light. In each exposure the excitation along a particular projection of the absorption dipole on the electric field leads to a distinct fluorescence intensity. Five exposures are sufficient to determine the full orientation of the fluorophores. To demonstrate the potential of the method we determine the orientation and position of individual immobilized lipid membrane markers. The shot-noise-limited isotropic angular resolution is 2°. For time-resolved studies the bandwidth can be expanded up to 200 Hz.

Incidence of metachronous contralateral inguinal hernias in children following unilateral repair — A meta-analysis of prospective studies

PURPOSE The objective of this review was to systematically evaluate the incidence of a metachronous contralateral inguinal hernia (MCIH) in children with unilateral inguinal hernia and therefore to propose or to reject routine contralateral groin exploration. METHODS Electronic searches restricted to prospective studies with a minimal follow-up of 1year included MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials. RESULTS Six studies involving 1669 children were included. Overall MCIH was 6% (95% CI from 4% to 8%). The odds for MCIH development were significantly larger in children with an initial left-sided hernia (OR 2.66 with 95% CI from 1.56 to 4.53) and in children with open contralateral processus vaginalis (CPV) (OR 4.17 with 95% CI from 1.25 to 13.9). CONCLUSIONS The overall incidence of MCIH following unilateral inguinal hernia repair in children is 6%. Initial left-sided hernia (8.5%) and open CPV (13.8%) are risk factors for MCIH development. Female gender (8.2%) and younger age (<1year) (6.9%) non-significantly increase the risk of MCIH.

Rapid adaptation to microgravity in mammalian macrophage cells

Despite the observed severe effects of microgravity on mammalian cells, many astronauts have completed long term stays in space without suffering from severe health problems. This raises questions about the cellular capacity for adaptation to a new gravitational environment. The International Space Station (ISS) experiment TRIPLE LUX A, performed in the BIOLAB laboratory of the ISS COLUMBUS module, allowed for the first time the direct measurement of a cellular function in real time and on orbit. We measured the oxidative burst reaction in mammalian macrophages (NR8383 rat alveolar macrophages) exposed to a centrifuge regime of internal 0 g and 1 g controls and step-wise increase or decrease of the gravitational force in four independent experiments. Surprisingly, we found that these macrophages adapted to microgravity in an ultra-fast manner within seconds, after an immediate inhibitory effect on the oxidative burst reaction. For the first time, we provided direct evidence of cellular sensitivity to gravity, through real-time on orbit measurements and by using an experimental system, in which all factors except gravity were constant. The surprisingly ultra-fast adaptation to microgravity indicates that mammalian macrophages are equipped with a highly efficient adaptation potential to a low gravity environment. This opens new avenues for the exploration of adaptation of mammalian cells to gravitational changes.

Single-Cell Phenotype Classification Using Deep Convolutional Neural Networks

Deep learning methods are currently outperforming traditional state-of-the-art computer vision algorithms in diverse applications and recently even surpassed human performance in object recognition. Here we demonstrate the potential of deep learning methods to high-content screening–based phenotype classification. We trained a deep learning classifier in the form of convolutional neural networks with approximately 40,000 publicly available single-cell images from samples treated with compounds from four classes known to lead to different phenotypes. The input data consisted of multichannel images. The construction of appropriate feature definitions was part of the training and carried out by the convolutional network, without the need for expert knowledge or handcrafted features. We compare our results against the recent state-of-the-art pipeline in which predefined features are extracted from each cell using specialized software and then fed into various machine learning algorithms (support vector machine, Fisher linear discriminant, random forest) for classification. The performance of all classification approaches is evaluated on an untouched test image set with known phenotype classes. Compared to the best reference machine learning algorithm, the misclassification rate is reduced from 8.9% to 6.6%.