Counting Fluorescent Dye Molecules on DNA Origami by Means of Photon Statistics

Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single‐molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1–20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well‐defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non‐invasively within 150 ms. The method might become a quantifying add‐on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT‐type microscopy.     

Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0. 77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.     

Test of a high resolution drift chamber prototype

The performance of a drift chamber prototype for a colliding beam vertex detector in a test beam at DESY is described. At one (two) atmosphere gas pressure a spatial resolution of 40 μm (30 μm) per wire for one cm drift length was achieved with a 100 MHz Flash-ADC system. An excellent double track resolution of better than 300 μm over the full drift length of 5 cm can be estimated.     

Hydraulischer Grundbruch: neue Erkenntnisse

Hydraulic heave: new findings. The contribution presents the results of investigations for the verification of hydraulic heave for excavations in front of walls with small embedment in the ground and with filter layers at the excavation side of the wall. For this case numerical groundwater flow computations show that there are significant vertical flow components below the wall which have to be considered in the verification of hydraulic heave. Based on the flow computation a procedure is presented to calculate the required height of the filter depending on the distance to the wall. This is compared with commonly used approaches. Furthermore, on the basis of this procedure, the integration of safety factors in accordance with DIN 1054 and of friction forces within the filter are explained.     

Therapeutic Treatments for Osteoporosis—Which Combination of Pills Is the Best among the Bad?

Osteoporosis is a chronical, systemic skeletal disorder characterized by an increase in bone resorption, which leads to reduced bone density. The reduction in bone mineral density and therefore low bone mass results in an increased risk of fractures. Osteoporosis is caused by an imbalance in the normally strictly regulated bone homeostasis. This imbalance is caused by overactive bone-resorbing osteoclasts, while bone-synthesizing osteoblasts do not compensate for this. In this review, the mechanism is presented, underlined by in vitro and animal models to investigate this imbalance as well as the current status of clinical trials. Furthermore, new therapeutic strategies for osteoporosis are presented, such as anabolic treatments and catabolic treatments and treatments using biomaterials and biomolecules. Another focus is on new combination therapies with multiple drugs which are currently considered more beneficial for the treatment of osteoporosis than monotherapies. Taken together, this review starts with an overview and ends with the newest approaches for osteoporosis therapies and a future perspective not presented so far.     

High resolution drift cells

We have built two test chambers, each 1 m long, containing quadratic and hexagonal cells which form approximately cylindrical drift fields. The chambers were filled with a gas mixture of carbon dioxide plus isobutan. They were read out with TDCs and with flash ADCs, alternatively. We arrive at space resolutions of 30 μm at atmospheric pressure and of ～ 25 μm at 2 bar pressure within the central part of the total drift length.     

A sequential sampling strategy for adaptive classification of computationally expensive data

Many real-world problems in engineering can be represented and solved as a data-driven classification problem, where the goal is to build a classifier that maps a given set of input parameters onto a corresponding class or label. In some cases, the collection of data samples can be computationally expensive. It is therefore crucial to solve the problem using as little data as possible. To this end, a novel sequential sampling algorithm is proposed that begins with a very small training set and supplements it in each iteration by a small batch of additional (expensive) data points. The outcome is a representative set of data samples that focuses the sampling on those locations in the input space where the class labels are changing more rapidly, while making sure that no class regions are missed.     

Imaging diffusion in living cells using time-correlated single-photon counting

Current efforts to monitor the diffusion of proteins in living cells are based on either fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching, or image correlation spectroscopy. However, these methods cannot generate a map of diffusion times. Here, we introduce a new method termed diffusion imaging microscopy that combines scanning confocal microscopy, time-correlated single-photon counting, and FCS and thus allows us to measure spatially resolved diffusion times. In our approach, we record scan images with time-resolved photon streams within each individual pixel. By extending the pixel dwell time to 25-100 ms, a software correlation of individual photons within each pixel yields the average diffusion time. Additionally, information on fluorescence intensity (number of photons) and fluorescence lifetime is available and can be used to sort fluorescence photons and to discriminate from autofluorescence. We evaluated our method by measuring diffusion times of dT20-TMR in solutions of different viscosity. We further demonstrate the applicability of the method to living cells and recorded a diffusion map of a living 3T3 mouse fibroblast incubated with dT20-ATTO488.