Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification

Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary 'blinking' without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation

Calculations of energy levels and lifetimes of low-lying states of barium and radium

We use the configuration interaction method and many-body perturbation theory to perform accurate calculations of energy levels, transition amplitudes, and lifetimes of low-lying states of barium and radium. Calculations for radium are needed for the planning of measurements of parity and time invariance violating effects which are strongly enhanced in this atom. Calculations for barium are used to control the accuracy of the calculations.Comment: 8 page

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super-resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy

Traveling-wave deceleration of SrF molecules

We report on the production, deceleration and detection of a SrF molecular beam. The molecules are captured from a supersonic expansion and are decelerated in the X$^2\Sigma^+ (v=0, N=1)$ state. We demonstrate the removal of up to 40% of the kinetic energy with a 2 meter long modular traveling-wave decelerator. Our results demonstrate a crucial step towards the preparation of ultracold gases of heavy diatomic molecules for precision spectroscopy

124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.We thank Martin Spitaler and the imaging facility of the MPI of Biochemistry for confocal imaging support

Lamb shift in muonic helium ion

The Lamb shift (2P_{1/2}-2S_{1/2}) in the muonic helium ion (mu ^4_2He)^+ is calculated with the account of contributions of orders alpha^3, alpha^4, alpha^5 and alpha^6. Special attention is given to corrections of the electron vacuum polarization, the nuclear structure and recoil effects. The obtained numerical value of the Lamb shift 1379.028 meV can be considered as a reliable estimate for the comparison with experimental data.Comment: 18 pages, 11 figure

Communication

Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single-molecule sensitivity. Here, we introduce Proximity-PAINT (pPAINT), a variation of the super-resolution microscopy technique DNA-PAINT. pPAINT uses a split-docking-site configuration to detect spatial proximity with high sensitivity, low false-positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha- and beta-tubulin in microtubules using super-resolution detection. © 2020 Wiley-VCH GmbH

Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems

Hormonal modifications in patients admitted to an internal intensive care unit for acute hypoxaemic respiratory failure

AbstractTo clarify which endocrine modifications can be observed in acute hypoxaemic respiratory failure, 15 severely ill male patients [PAT; median age: 61 (range: 48 years); median height: 173 (range: 12) cm; median mass: 73 (range 31) kg] were investigated immediately upon admission to an intensive care unit (ICU) for this clinical disorder. Before starting treatment, the blood gases were measured and a number of selected hormones with special relevance for an ICU setting were determined. These are known to be modified by acute hypoxaemia in healthy subjects and to possess glucoregulatory properties, or an influence upon cardiocirculation or the vascular volume regulation: insulin, cortisol, adrenaline, noradrenaline, atrial natriuretic peptide, renin, aldosterone, angiotensin converting enzyme, and endothelin-I (ET). To elucidate whether potential endocrine changes resulted from acute hypoxaemia alone, the underlying disease, or unspecific influences connected with the ICU setting, all measurements were compared to those of a completely healthy reference group (REF) with comparable acute experimental hypoxaemia. The latter state was achieved by having the REF breathe a gas mixture with the oxygen content reduced to 14% (H).In the REF, neither the medians nor the distribution of endocrinologic measurements were modified significantly by acute hypoxaemia. In the PAT, the medians were increased considerably, yet with a slight diminution of ET. The distribution of individual values was considerably broader than in the REF with H.In conclusion, considerable increases in the means of the above hormones, with the exception of ET, can be registered in severely ill patients admitted to ICUs with acute hypoxaemic failure. However, such modifications cannot be considered attributable exclusively to acute arterial hypoxaemia. The underlying clinical disorders, such as septicaemia or an unspecific endocrine epiphenomenon, including severe and not only hypoxaemic stress, seem to be predominant