4-Aminobenzoic acid, 2-phenoxyethanol and iodine used as tracers in a short-term in vivo-kinetics study for tattoo ink ingredients: Mass spectrometry method development and validation.

Tattoos have been gaining popularity in recent years, leading to a growing interest in researching tattoo inks and the tattooing process itself. Since the exposure to soluble tattoo ink ingredients has not yet been investigated, we here present the method validation for a short-term biokinetics study on soluble tattoo ink ingredients. The three tracers 4-aminobenzoic acid (PABA), 2-phenoxyethanol (PEtOH) and iodine will be added to commercially available tattoo inks, which will subsequently be used on healthy study participants. Following the tattooing process, blood and urine will be sampled at specific time points and analysed for these tracers. For this purpose, a method using liquid chromatography separation coupled to a quadrupole time-of-flight mass spectrometer (LC-QTOF-MS) in positive and negative ESI mode for the quantification of PABA, PEtOH and selected metabolites and an inductively-coupled plasma (ICP)-MS method for the determination of iodine were developed and validated. For LC-QTOF-MS analysis, the most applicable additives for LC eluents (0.01 % formic acid for positive and 0.005 % acetic acid for negative mode) were identified. Protein precipitation with acetonitrile was chosen for sample preparation. The methods were validated for selectivity, specificity, carryover, linearity, limit of detection (LOD) and quantification (LOQ), matrix effects, accuracy and precision, stability under different conditions and dilution integrity according to national and international guidelines with an allowed maximum variation of ±15 %. The LC-QTOF-MS method met the imposed guideline criteria for most parameters, however, some metabolites showed strong matrix effects. Validation of the ICP-MS method revealed that the KED-H2 collision mode is superior to the standard analysis mode due to enhanced method accuracy. The methods were validated for the relevant matrices plasma, urine, tattoo ink and tattoo consumables and proved to be applicable for the main target substances in the short-term biokinetics study. A proof-of-concept study showed successful quantification of iodine and PABA metabolites. The PEtOH metabolite was also quantified, but showed strong matrix effects in urine. Therefore standard addition was selected as an alternative quantification method.

Peat and disinfectant powder used in swine husbandry systems - quantification of oral intake using toxic metals as potential markers.

The use of enrichment and bedding materials in pig husbandry intends to comply with the animals' behavioural needs to perform natural exploratory behaviour, which is strongly connected to foraging behaviour. It can thus be assumed that pigs will ingest a certain material quantity possibly posing a risk to animal health and food safety as previous studies identified contaminants in enrichment and bedding materials. However, risk assessment requires knowledge about the effective amount of ingested material. Voluntary material intake of pigs with free access to peat and disinfectant powder was estimated by measuring the tissue levels of toxic metals originating from the respective materials in 28 pigs (seven groups, n = 4) via inductively coupled plasma mass spectrometry and comparing the results to tissue levels of pigs fed with known amounts of metals. Additionally, as markers of consumption, n-alkanes and acid insoluble ash naturally occurring in the materials and titanium dioxide, added as an external marker to disinfectant powder, were analysed in pigs' faeces. Tissue levels of toxic metals as well as marker analyses in pigs' faeces could prove material consumption. Results revealed mean voluntary intake levels of peat and disinfectant powder by pigs up to 7% and 2% of the daily ration. Hence, a transfer of contained toxic metals into the food chain might occur. Although current maximum levels for toxic elements in animal tissues were not exceeded due to dietary inclusion of peat or disinfectant powder, dietary exposure through food of animal origin should be reduced to a possible minimum. This applies specifically for elements, where no health-based guidance values for humans could have been derived (e.g. arsenic). Thus, labelling guidelines for enrichment and bedding materials can be a perspective to limit the entry of toxic metals and trace elements into the environment.

MINFLUX imaging of a bacterial molecular machine at nanometer resolution.

The resolution achievable with the established super-resolution fluorescence nanoscopy methods, such as STORM or STED, is in general not sufficient to resolve protein complexes or even individual proteins. Recently, minimal photon flux (MINFLUX) nanoscopy has been introduced that combines the strengths of STED and STORM nanoscopy and can achieve a localization precision of less than 5 nm. We established a generally applicable workflow for MINFLUX imaging and applied it for the first time to a bacterial molecular machinein situ, i.e., the injectisome of the enteropathogenY. enterocolitica. We demonstrate with a pore protein of the injectisome that MINFLUX can achieve a resolution down to the single molecule levelin situ. By imaging a sorting platform protein using 3D-MINFLUX, insights into the precise localization and distribution of an injectisome component in a bacterial cell could be accomplished. MINFLUX nanoscopy has the potential to revolutionize super-resolution imaging of dynamic molecular processes in bacteria and eukaryotes.

A pilot study on the nanoscale properties of bone tissue near lacunae in fracturing women.

The goal of this study is to investigate the causes of osteoporosis-related skeletal fragility in postmenopausal women. We hypothesize that bone fragility in these individuals is largely due to mineral, and/or intrinsic material properties in the osteocyte lacunar/peri-lacunar regions of bone tissue. Innovative measurements with nanoscale resolution, including scanning electron microscope (SEM), an atomic force microscope that is integrated with infrared spectroscopy (AFM-IR), and nanoindentation, were used to characterize osteocyte lacunar and peri-lacunar properties in bone biopsies from fracturing (Cases) and matched (Age, BMD), non-fracturing (Controls) postmenopausal healthy women. In the peri-lacunar space, the nanoindentation results show that the modulus and hardness of the Controls are lower than the Cases. The AFM-IR results conclusively show that the mineral matrix, maturity (peak) (except in outer/far regions in Controls) were greater in Controls than in Cases. Furthermore, these results indicate that while mineral-to-matrix area ratio tend to be greater, the mineral maturity and crystallinity peak ratio "near" lacunae is greater than at regions "far" or more distance from lacunae in the Controls only. Due to the heterogeneity of bone structure, additional measurements are needed to provide more convincing evidence of altered lacunar characteristics and changes in the peri-lacunar bone as mechanisms related to postmenopausal women and fragility. Such findings would motivate new osteocyte-targeted treatments to reduce fragility fracture risks in these groups.

Resolving the molecular architecture of the photoreceptor active zone with 3D-MINFLUX.

Cells assemble macromolecular complexes into scaffoldings that serve as substrates for catalytic processes. Years of molecular neurobiology research indicate that neurotransmission depends on such optimization strategies. However, the molecular topography of the presynaptic active zone (AZ), where transmitter is released upon synaptic vesicle (SV) fusion, remains to be visualized. Therefore, we implemented MINFLUX optical nanoscopy to resolve the AZ of rod photoreceptors. This was facilitated by a novel sample immobilization technique that we name heat-assisted rapid dehydration (HARD), wherein a thin layer of rod synaptic terminals (spherules) was transferred onto glass coverslips from fresh retinal slices. Rod ribbon AZs were readily immunolabeled and imaged in 3D with a precision of a few nanometers. Our 3D-MINFLUX results indicate that the SV release site in rods is a molecular complex of bassoon-RIM2-ubMunc13-2-Cav1.4, which repeats longitudinally on both sides of the ribbon.

MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope.

The recently introduced minimal photon fluxes (MINFLUX) concept pushed the resolution of fluorescence microscopy to molecular dimensions. Initial demonstrations relied on custom made, specialized microscopes, raising the question of the method's general availability. Here, we show that MINFLUX implemented with a standard microscope stand can attain 1-3 nm resolution in three dimensions, rendering fluorescence microscopy with molecule-scale resolution widely applicable. Advances, such as synchronized electro-optical and galvanometric beam steering and a stabilization that locks the sample position to sub-nanometer precision with respect to the stand, ensure nanometer-precise and accurate real-time localization of individually activated fluorophores. In our MINFLUX imaging of cell- and neurobiological samples, ~800 detected photons suffice to attain a localization precision of 2.2 nm, whereas ~2500 photons yield precisions <1 nm (standard deviation). We further demonstrate 3D imaging with localization precision of ~2.4 nm in the focal plane and ~1.9 nm along the optic axis. Localizing with a precision of <20 nm within ~100 µs, we establish this spatio-temporal resolution in single fluorophore tracking and apply it to the diffusion of single labeled lipids in lipid-bilayer model membranes.

Characterisation of Elastomers as Food Contact Materials-Part 1: Quantification of Extractable Compounds, Swelling of Elastomers in Food Simulants and Release of Elements.

Elastomers are not a uniform class of materials but comprise a broad spectrum of chemically different polymers. Sealing gaskets, gloves, teats, conveyor belts and tubing are examples of elastomers being used as food contact materials (FCMs). Ten elastomer samples were evaluated with respect to the content of extractable compounds, migration of substances into ethanolic food simulants, swelling in food simulants and release of elements in different food simulants. The number of extractable substances <1000 Da was determined by comprehensive two-dimensional gas chromatography coupled with flame ionisation detection (GC × GC-FID) analysis of tetrahydrofuran (THF) extracts. The number of signals ranged from 61 (a thermoplastic elastomer (TPE)) to 690 (a natural rubber/styrene-butadiene-rubber blend (NR/SBR)). As for risk assessment, the decisive factor is which substances reach the food. The extent of substances that migrate into ethanolic food simulants was investigated. Elastomer FCMs can be the source of food contamination with heavy metals. Notably, contamination with lead was detected in some samples investigated in this study. It was shown that food simulants harbour the potential to morphologically alter or even disintegrate elastomeric materials. The results presented here highlight the importance to carefully choose the elastomer type for the intended use as FCMs as not every application may prove safe for consumers.

Release of aluminium and thallium ions from uncoated food contact materials made of aluminium alloys into food and food simulant.

In order to investigate the release of aluminium ions from food contact materials, three different types of uncoated aluminium menu trays for single use were tested with the foodstuffs sauerkraut juice, apple sauce and tomato puree, as well as with the food simulants 5 g/L citric acid solution and artificial tap water. To mimic a consumer relevant exposure scenario, the aluminium trays were studied using time and temperature gradients according to the Cook & Chill method, also taking into account storage time at elevated temperatures during the delivery period. The release of aluminium was found to exceed the specific release limit (SRL) of 5 mg aluminium per kilogram of food specified by the Council of Europe by up to six times. Furthermore, a release of thallium was also detected unexpectedly. Kinetic studies showed a comparable behaviour in the release of aluminium, manganese and vanadium as components of the aluminium alloy itself. In contrast, thallium could be identified as a surface contaminant or impurity because of an entirely different kinetic curve. Kinetic studies also allowed activation energy calculations. Additional camping saucepans were tested as an article for repeated use. In three subsequent release experiments with citric acid (5 g/L), artificial tap water and tomato puree as benchmark foodstuffs, the results were comparable to those of the uncoated wrought alloy aluminium trays.

4Pi-RESOLFT nanoscopy.

By enlarging the aperture along the optic axis, the coherent utilization of opposing objective lenses (4Pi arrangement) has the potential to offer the sharpest and most light-efficient point-spread-functions in three-dimensional (3D) far-field fluorescence nanoscopy. However, to obtain unambiguous images, the signal has to be discriminated against contributions from lobes above and below the focal plane, which has tentatively limited 4Pi arrangements to imaging samples with controllable optical conditions. Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution.

isoSTED nanoscopy with intrinsic beam alignment.

Despite the need for isotropic optical resolution in a growing number of applications, the majority of super-resolution fluorescence microscopy setups still do not attain an axial resolution comparable to that in the lateral dimensions. Three-dimensional (3D) nanoscopy implementations that employ only a single objective lens typically feature a trade-off between axial and lateral resolution. 4Pi arrangements, in which the sample is illuminated coherently through two opposing lenses, have proven their potential for rendering the resolution isotropic. However, instrument complexity due to a large number of alignment parameters has so far thwarted the dissemination of this approach. Here, we present a 4Pi-STED setup combination, also called isoSTED nanoscope, where the STED and excitation beams are intrinsically co-aligned. A highly robust and convenient 4Pi cavity allows easy handling without the need for readjustments during imaging experiments.

Metal release from coffee machines and electric kettles.

The release of elemental ions from 8 coffee machines and 11 electric kettles into food simulants was investigated. Three different types of coffee machines were tested: portafilter espresso machines, pod machines and capsule machines. All machines were tested subsequently on 3 days before and on 3 days after decalcification. Decalcification of the machines was performed with agents according to procedures as specified in the respective manufacturer's manuals. The electric kettles showed only a low release of the elements analysed. For the coffee machines decreasing concentrations of elements were found from the first to the last sample taken in the course of 1 day. Metal release on consecutive days showed a decreasing trend as well. After decalcification a large increase in the amounts of elements released was encountered. In addition, the different machine types investigated clearly differed in their extent of element release. By far the highest leaching, both quantitatively and qualitatively, was found for the portafilter machines. With these products releases of Pb, Ni, Mn, Cr and Zn were in the range and beyond the release limits as proposed by the Council of Europe. Therefore, a careful rinsing routine, especially after decalcification, is recommended for these machines. The comparably lower extent of release of one particular portafilter machine demonstrates that metal release at levels above the threshold that triggers health concerns are technically avoidable.

Waterpipe smoke: source of toxic and carcinogenic VOCs, phenols and heavy metals?

The use of the waterpipe, a traditional aid for the consumption of tobacco, has spread worldwide and is steadily increasing especially among the youth. On the other hand, there is a lack of knowledge regarding the composition of mainstream waterpipe smoke and the toxicological risks associated with this kind of smoking habit. Using a standardized machine smoking protocol, mainstream waterpipe smoke was generated and further analyzed for twelve volatile organic compounds (VOCs) and eight phenolic compounds by applying gas chromatography-mass spectrometry and reverse-phase high-performance liquid chromatography-fluorescence detection, respectively. Additionally, seventeen elements were analyzed in waterpipe tobacco and charcoal prior to and after smoking, applying inductively coupled plasma-mass spectrometry to assess the maximum exposure of these elements. For the first time ever, we have been able to show that waterpipe mainstream smoke contains high levels of the human carcinogen benzene. Compared with cigarette smoke yields, the levels were 6.2-fold higher, thus representing a significant health hazard for the waterpipe smoker. Furthermore, we found that waterpipe mainstream smoke contains considerable amounts of catechol, hydroquinone and phenol, each of which causing some health concern at least. The analysis of waterpipe tobacco and charcoal revealed that both matrices contained considerable amounts of the toxic elements nickel, cadmium, lead and chromium. Altogether, the data on VOCs, phenols and elements presented in this study clearly point to the health hazards associated with the consumption of tobacco using waterpipes.

4Pi Microscopy.

Optical microscopy has become a key technology in the life sciences today. Its noninvasive nature provides access to the interior of intact and even living cells, where specific molecules can be precisely localized by fluorescent tagging. However, the attainable 3D resolution of an optical microscope has long been hampered by a comparatively poor resolution along the optic axis. By coherent focusing through two objective lenses, 4Pi microscopy improves the axial resolution by three- to fivefold. This primer is intended as a starting point for the design and operation of a 4Pi microscope of type A.

Formation of nanopore-spanning lipid bilayers through liposome fusion.

Self-assembly of nanopore-spanning lipid bilayers (npsLBs) paves the way toward chip-based integrated membrane protein biosensing. We present a novel approach to analyze the formation of npsLB at individual nanopores using quantitative analysis of high-resolution microscopy images. From this analysis we derive necessary conditions for the formation of npsLBs on nanopore arrays by liposome fusion and discuss the limitations of the process as a function of nanopore geometry, lipid membrane properties, and surface interaction. Most importantly, applying liposomes with diameters larger than the nanopore is demonstrated to be a necessary but not sufficient condition for npsLB formation. A theoretical model is used to discuss and explain this experimental finding.

A readily retrievable pool of synaptic vesicles.

Although clathrin-mediated endocytosis is thought to be the predominant mechanism of synaptic vesicle recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a presorted and preassembled pool of synaptic vesicle proteins on the presynaptic membrane might support a first wave of fast clathrin-mediated endocytosis. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of synaptic vesicle proteins in rat hippocampal neurons using a new type of probe. By applying cypHer5E, a new cyanine dye-based pH-sensitive exogenous marker, coupled to antibodies to luminal domains of synaptic vesicle proteins, we could reliably monitor synaptic vesicle recycling and demonstrate the preferential recruitment of a surface pool of synaptic vesicle proteins upon stimulated endocytosis. By using fluorescence nanoscopy of surface-labeled synaptotagmin 1, we could resolve the spatial distribution of the surface pool at the periactive zone in hippocampal boutons, which represent putative sites of endocytosis.

A few immobilized thrombins are sufficient for platelet spreading.

Eukaryotic cells respond to signaling molecules with picomolar to nanomolar sensitivities. However, molar concentrations give no suggestion of the sufficient number of molecules per cell and are confusing when referring to physiological situations in which signaling molecules act in an immobilized state. Here, we studied platelet adhesion by thrombin, a key step in normal hemostasis and pathological arterial thrombosis. We generated a biofunctional nanosheet surface to mimic the in vivo solid-state interaction between platelets and thrombin at sites of injured tissues. We observed that <10 molecules readily activate platelets with high specificity, resulting in platelet adhesion and spreading. This number is much lower than expected from previous experiments in solution, in which the sole activation of platelets required a >1000-fold stoichiometric excess of thrombin. We conclude that immobilizing thrombin apposed to the membrane receptor allows platelets to respond with very high sensitivity. Moreover, we propose that irreversible cell activation may require several ligands to avoid activation by single, mislocalized signaling molecules.

Automatic deconvolution in 4Pi-microscopy with variable phase.

4Pi-microscopy doubles the aperture of the imaging system by coherent addition of the wavefronts for illumination and/or detection through opposing objective lenses. This improves the axial resolution 3-7 fold, but the raw data usually features ghost images which have to be removed by image reconstruction. This straightforward procedure is sometimes precluded by imperfect alignment of the instrument or a specimen with strong variations of its refractive index, because the image formation process now depends on the space-variant phase difference between the counter-propagating wavefronts. Here we present a computationally fast method of parametric blind deconvolution that allows for automatic and robust simultaneous estimation of both the object and the phase function in such cases. We verify the performance of our approach on both synthetic and real data. Because the method does not require a-priori knowledge of the phase function it is major step towards reliable 4Pi-imaging and automatic image restoration by non-expert users.

Sample preparation for STED microscopy.

Since the discovery of the diffraction barrier in the late nineteenth century, it has been commonly accepted that with far-field optical microscopy it is not possible to resolve structural details considerably finer than half the wavelength of light. The emergence of STED microscopy showed that, at least for fluorescence imaging, these limits can be overcome. Since STED microscopy is a far-field technique, in principle, the same sample preparation as for conventional confocal microscopy may be utilized. The increased resolution, however, requires additional precautions to ensure the structural preservation of the specimen. We present robust protocols to generate test samples for STED microscopy. These protocols for bead samples and immunolabeled mammalian cells may be used as starting points to adapt existing labeling strategies for the requirements of sub-diffraction resolution microscopy.

Mitochondrial cristae revealed with focused light.

Because of the diffraction resolution barrier, optical microscopes have so far failed in visualizing the mitochondrial cristae, that is, the folds of the inner membrane of this 200 to 400 nm diameter sized tubular organelle. Realizing a approximately 30 nm isotropic subdiffraction resolution in isoSTED fluorescence nanoscopy, we have visualized these essential structures in the mitochondria of intact cells. We find a pronounced heterogeneity in the cristae arrangements even within individual mitochondrial tubules.