New catalytic materials for the high-temperature synthesis of hydrocyanic acid from methane and ammonia by high-throughput approach

For converting methane and ammonia to hydrocyanic acid, catalysts were prepared and tested in a 48-parallel channel fixed-bed reactor unit operating at temperatures up to 1373 K. The catalysts were synthesized with a robot applying a genetic algorithm as the design tool. New and improved catalyst compositions were discovered by using a total of seven generations each consisting of 92 potential catalysts. Thereby, the catalyst support turned out as an important input variable. Furthermore, platinum, which is well known as a catalytic material was confirmed. Moreover, improvements in HCN yield were achieved by addition of promoters like Ir, Au, Ni, Mo, Zn and Re. Multi-way analysis of variance and regression trees were applied to establish correlations between HCN yield and catalyst composition (support and metal additives). The obtained results are considered as the base for future even more efficient screening experiments.     

Topological photoaffinity labeling of the rabbit ileal Na+/bile-salt-cotransport system

For the investigation of the topology of the rabbit ileal Na+/bile-salt-cotransport system, composed of a 93-kDa integral membrane protein and a peripheral 14-kDa bile-acid-binding protein (ILBP), we have synthesized photolabile dimeric bile-salt-transport inhibitors (photoblockers), G1-X-G2, where two bile acid moieties (G1 and G2) are tethered together via a spacer, X, and where one of the two bile acid moieties carries a photoactivatable group. These photoblockers specifically interact with the ileal Na+/bile-salt-cotransport system as demonstrated by a concentration-dependent inhibition of [3H]cholyltaurine uptake by rabbit ileal brush-border membrane vesicles and by inhibition of photolabeling of the 93-kDa and 14-kDa bile-salt-binding proteins by 7,7-azo and 3,3-azo derivatives of cholyltaurine. Ileal bile-salt uptake was specifically inhibited by the photoblockers, which were not taken up themselves by the small intestine as demonstrated by in vivo ileal perfusion. Dependent on the photoblocker used several polypeptides in the molecular-mass range of 14-130 kDa were labeled. The cytoplasmically attached 14-kDa ILBP was significantly labeled only by inhibitors that are photoactivatable in bile acid moiety G1, suggesting that during binding and translocation of a bile-salt molecule by the ileal bile-salt-transport system the steroid nucleus gets access to the cytoplasmic site of the ileal brush-border membrane first. Photoaffinity labeling in the frozen state with the transportable 3,3-azo and 7,7-azo derivatives of cholyltaurine revealed a time-dependent increase in the extent of labeling of the 14-kDa and 93-kDa proteins, suggesting a labeling of these proteins from the cytoplasmic site of the ileal brush-border membrane. By photoaffinity labeling in the frozen state with the various photoblockers time-dependent changes in the extent of photoaffinity labeling of bile-salt-binding proteins were observed, demonstrating the possibility of topological analysis of the rabbit ileal Na+/bile-salt-cotransport system.     

99mTc-HDP Labeling—A Non-Destructive Method for Real-Time Surveillance of the Osteogenic Differentiation Potential of hMSC during Ongoing Cell Cultures

99-Metastabil Technetium (^99mTc) is a radiopharmaceutical widely used in skeletal scintigraphy. Recent publications show it can also be used to determine the osteogenic potential of human mesenchymal stem cells (hMSCs) by binding to hydroxyapatite formed during bone tissue engineering. This field lacks non-destructive methods to track live osteogenic differentiation of hMSCs. However, no data about the uptake kinetics of ^99mTc and its effect on osteogenesis of hMSCs have been published yet. We therefore evaluated the saturation time of ^99mTc by incubating hMSC cultures for different periods, and the saturation concentration by using different amounts of ^99mTc activity for incubation. The influence of ^99mTc on osteogenic potential of hMSCs was then evaluated by labeling a continuous hMSC culture three times over the course of 3 weeks, and comparing the findings to cultures labeled once. Our findings show that ^99mTc saturation time is less than 0.25 h, and saturation concentration is between 750 and 1000 MBq. Repeated exposure to γ-radiation emitted by ^99mTc had no negative effects on hMSC cultures. These new insights can be used to make this highly promising method broadly available to support researchers in the field of bone tissue engineering using this method to track and evaluate, in real-time, the osteogenic differentiation of hMSC, without any negative influence on the cell viability, or their osteogenic differentiation potential.     

Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching

Subdiffraction fluorescence imaging is presented in a parallelized wide-field arrangement exploiting the principle of reversible saturable/switchable optical transitions (RESOLFT). The diffraction barrier is overcome by photoswitching ensembles of the label protein asFP595 between a nonfluorescent off- and a fluorescent on-state. Relying on ultralow continuous-wave intensities, reversible protein switching facilitates parallelized fast image acquisition. The RESOLFT principle is implemented by illuminating with intensity distributions featuring zero intensity lines that are further apart than the conventional Abbe resolution limit. The subdiffraction resolution is verified by recording live Escherichia coli bacteria labeled with asFP595. The obtained resolution of 50 nm ( approximately lambda/12) is limited only by the spectroscopic properties of the proteins and the imperfections of the optical implementation, but not on principle grounds.     

A new high-aperture glycerol immersion objective lens and its application to 3D-fluorescence microscopy

High‐resolution light microscopy of glycerol‐mounted biological specimens is performed almost exclusively with oil immersion lenses. The reason is that the index of refraction of the oil and the cover slip of ~1.51 is close to that of ~1.45 of the glycerol mountant, so that refractive index mismatch‐induced spherical aberrations are tolerable to some extent. Here we report the application of novel cover glass‐corrected glycerol immersion lenses of high numerical aperture (NA) and the avoidance of these aberrations. The new lenses feature a semi‐aperture angle of 68.5°, which is slightly larger than that of the diffraction‐limited 1.4 NA oil immersion lenses. The glycerol lenses are corrected for a quartz cover glass of 220 µm thickness and for a 80% glycerol‐water immersion solution. Featuring an aberration correction collar, the lens can adapt to glycerol concentrations ranging between 72% and 88%, to slight variations of the temperature, and to the cover glass thickness. As the refractive index mismatch‐induced aberrations are particularly important to quantitative confocal fluorescence microscopy, we investigated the axial sectioning ability and the axial chromatic aberrations in such a microscope as well as the image brightness as a function of the penetration depth. Whereas there is a significant decrease in image brightness associated with oil immersion, this decrease is absent with the glycerol immersion system. In addition, we show directly the compression of the optic axis in the case of oil immersion and its absence in the glycerol system. The unique advantages of these new lenses in high‐resolution microscopy with two coherently used opposing lenses, such as 4 Pi‐microscopy, are discussed.     

Three-dimensional tracking of single fluorescent particles with submillisecond temporal resolution

Observing dynamics at the nanoscale requires submillisecond time resolution. Notably, in studying biological systems, three-dimensional (3D) trajectories of fluorescently labeled objects such as viruses or transport vesicles often need to be acquired with high temporal resolution. Here, we present a novel instrument that combines scanning-free multiplane detection at 3.2 kHz frame rate and single photon sensitivity with optimized beam-steering capabilities. This setup enables ultrafast 3D localization with submillisecond time resolution and nanometer localization precision. We demonstrate 3D tracking of single fluorescent particles at speeds of up to 150 nm/ms over several seconds and large volumes. By focused excitation of only the particle of interest, while avoiding confocal pinholes, the system realizes maximum detection efficiency at minimal laser irradiation. These features, combined with the avoidance of stage movement, provide high live-sample compatibility for future biomedical applications.     

Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz

We show the viability of high-resolution two-photon fluorescence imaging of fixed and live cells by exciting the fluorophores with a train of near-infrared pulses with duration in the picosecond range. This is exemplified with a compact, diode-pumped Nd:YVO₄ laser, emitting trains of 7-ps pulses at a wavelength of 1064 nm, with a repetition rate of 200 MHz at two separate outputs. Incoherent combination of the outputs enabled two-photon excitation with a repetition rate of 400 MHz. For a numerical aperture of 1.4 (oil), we used an average illumination power of up to 20–40 mW at the sample. The pulses were coupled into a beam scanning microscope, either directly or through a single mode glass fibre. Compared with standard femtosecond titanium–sapphire excitation conditions, our experiments were performed with a 2.5 or 5 times higher repetition rate, 30–70 times longer pulses and 10–35 times lower pulse peak intensity. The experiments indicate the possibility of significantly relaxing the temporal pulse width constraints for a series of applications.     

Structural studies of the H+/oligopeptide transport system from rabbit small intestine

A 127-kDa protein was identified as a component of the H+/oligopeptide transport system in brush-border membrane vesicles from rabbit small intestine by photoaffinity labeling with [3H]cephalexin and further photoreactive beta-lactam antibiotics and dipeptides. Reconstitution of stereospecific transport activity revealed the involvement of the 127-kDa protein in H+-dependent transport of oligopeptides and orally active alpha-amino-beta-lactam antibiotics (Kramer et al., Eur. J. Biochem. 204 (1992) 923-930). H+-Dependent transport activity was found in all segments of the small intestine concomitantly with the specific labeling of the 127-kDa protein. By enzymatic deglycosylation, fragments of Mr 116 and 95 kDa were obtained from the 127-kDa protein with endoglucosidase F and N-glycanase, whereas with endoglucosidase H, a fragment of Mr 116 kDa was formed. These findings indicate that the photolabeled 127-kDa protein is a microheterogenous glycoprotein. Surprisingly, it was found that the solubilized and purified 127-kDa protein showed enzymatic sucrase and isomaltase activity. Inhibition of the glucosidase activities with the glucosidase inhibitor HOE 120 influenced neither H+/oligopeptide transport nor photoaffinity labeling of the 127-kDa protein. With polyclonal antibodies raised against the purified 127-kDa protein, a coprecipitation of sucrase activity and the photolabeled 127-kDa beta-lactam antibiotic binding protein occurred. Target size analysis revealed a functional molecular mass of 165+/-17 kDa for photoaffinity labeling of the 127-kDa protein, suggesting a homo- or heterodimeric functional structure of the 127-kDa protein in the brush-border membrane. These findings indicate that the H+/oligopeptide binding protein of Mr 127000 is closely associated with the sucrase/isomaltase complex in the enterocyte brush-border membrane.