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BOOK
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.
Minimal photon fluxes (MINFLUX) has enabled molecule-scale resolution in fluorescence microscopy but this had not been shown in standard, broadly applicable microscopy platforms. Here the authors report a solution to allow normal fluorescence microscopy while also providing 1-3 nm 3D resolution.
Journal Article
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.
The ability to discriminate objects along the optic axis is a benchmark for three-dimensional imaging techniques. Here, the authors combine metastable-state switching and opposing objective lenses to suppress out-of-focus background and record three-dimensional nanoscale images of living cells.
Journal Article
Spherical nanosized focal spot unravels the interior of cells
A fluorescence microscope relying entirely on focused light allows the generation of spherical focal fluorescence spots much smaller than the wavelength of light. This development, termed isoSTED, overcomes the resolution limitation imposed by the diffraction of light and permits three-dimensional nanoscale imaging inside cells with common fluorophores.
The resolution of any linear imaging system is given by its point spread function (PSF) that quantifies the blur of an object point in the image. The sharper the PSF, the better the resolution is. In standard fluorescence microscopy, however, diffraction dictates a PSF with a cigar-shaped main maximum, called the focal spot, which extends over at least half the wavelength of light (
λ
= 400–700 nm) in the focal plane and >
λ
along the optical axis (
z
). Although concepts have been developed to sharpen the focal spot both laterally and axially, none of them has reached their ultimate goal: a spherical spot that can be arbitrarily downscaled in size. Here we introduce a fluorescence microscope that creates nearly spherical focal spots of 40–45 nm (
λ
/16) in diameter. Fully relying on focused light, this lens-based fluorescence nanoscope unravels the interior of cells noninvasively, uniquely dissecting their sub-
λ
–sized organelles.
Journal Article
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.
Journal Article
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.
Journal Article
Diffraction-unlimited three-dimensional optical nanoscopy with opposing lenses
The resolution of far-field optical microscopy stagnated for a century, but a quest began in the 1990s leading to nanoscale imaging of transparent fluorescent objects in three dimensions. Important elements in this pursuit were the synthesis of the aperture of two opposing lenses and the modulation or switching of the fluorescence of adjacent markers. The first element provided nearly isotropic three-dimensional resolution by improving the axial resolution by three- to sevenfold, and the second enabled the diffraction barrier to be overcome. Here, we review recent progress in the synergistic combination of these two elements which non-invasively provide an isotropic diffraction-unlimited three-dimensional resolution in transparent objects.
Journal Article
Information sharing versus order aggregation strategies in supply chains
Purpose - The purpose of this paper is to explore how differently aggregated order data may affect inventories and service levels in a serial supply chain and compares the results against various levels of information sharing. By performing sensitivity analysis, critical parameters are identified and conjectures for explaining the divergent results on the value of information sharing in prior literature are given.Design methodology approach - By using discrete event simulation, the paper analyses various approaches of differently aggregated order data compared to shared demand information.Findings - The experiments show that suppliers cannot accurately estimate demand means and variances because of time-depending order quantities and biasing effects of order inter-arrival times. This may lead to inappropriate computations of reorder points and safety stocks. The aggregation of order data can improve the calculations resulting in lower inventories with almost identical service levels. The mean inventory can also be reduced by sharing information but may lead to considerably lower service levels.Research limitations implications - As discovered in this paper, simplifications in the supply chain structure may have large effects on the experimental results. Therefore, the value of information sharing and order aggregation strategies should be analyzed in a more complex supply chain network.Practical implications - Some ordering mechanisms have the effect of increasing the demand variance for upstream companies. This amplification may lead to inefficiencies throughout the entire supply chain. The paper proposes solutions to managers on how they can benefit from order data aggregation and information sharing. The per period variances may be reduced leading to smaller safety stocks and lower costs for the entire supply chain.Originality value - The paper shows that the performance of a supply chain may be improved by aggregating order data and compares the results with improvements derived from information sharing strategies.
Journal Article
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.
Journal Article