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Abstract Utilizing flow cytometry to monitor progress of bulk biochemical reactions and concentration of chemical species normally relies on the utilization of cells carrying intrinsic fluorescence or modified beads. We present a method for a simple measurement of the fluorescent marker molecule fluorescein and GFPuv in bulk solutions with high sensitivity using a CytoFLEX flow cytometer and without the need for modified beads. Polystyrene beads were used to trigger measurements based on their high scatter signal, to detect the fluorescence signal from two different fluorophores present in the sample solution. We report sensitivities of 33 pg/mL for fluorescein and 50 ng/mL for GFPuv. This method is comparable in sensitivity to a typical spectrometric fluorescence assay tested with fluorescein, and approximately ten times more sensitive for the measurement of GFPuv. PEG was added to the sample at a low concentration of 0.001% (w/v) to block unspecific GFPuv binding to the beads. The method was further applied to measure the GFPuv concentration in crude cell lysate samples used for cell free protein expression. An advantage of this method over spectrometric assays is the ability to differentiate signal subpopulations in the sample based on their individual fluorescence intensities. Highlights • Simple measurement of fluorophores in bulk solutions using flow cytometry. • Non-specifically prepared beads used for triggering. • Sensitivity (GFPuv: ~50 ng/mL) is ten times better than spectrometric assay. • Applicable also in crude cell lysate as used for cell free protein expression. [ABSTRACT FROM AUTHOR]

Background: With additive manufacturing (AM) individual and biocompatible implants can be generated by using suitable materials. The aim of this study was to investigate the biological effects of polylactic acid (PLA) manufactured by Fused Deposition Modeling (FDM) on osteoblasts in vitro according to European Norm / International Organization for Standardization 10,993-5. Method: Human osteoblasts (hFOB 1.19) were seeded onto PLA samples produced by FDM and investigated for cell viability by fluorescence staining after 24 h. Cell proliferation was measured after 1, 3, 7 and 10 days by cell-counting and cell morphology was evaluated by scanning electron microscopy. For control, we used titanium samples and polystyrene (PS). Results: Cell viability showed higher viability on PLA (95,3% ± 2.1%) than in control (91,7% ±2,7%). Cell proliferation was highest in the control group (polystyrene) and higher on PLA samples compared to the titanium samples. Scanning electron microscopy revealed homogenous covering of sample surface with regularly spread cells on PLA as well as on titanium. Conclusion: The manufacturing of PLA discs from polylactic acid using FDM was successful. The in vitro investigation with human fetal osteoblasts showed no cytotoxic effects. Furthermore, FDM does not seem to alter biocompatibility of PLA. Nonetheless osteoblasts showed reduced growth on PLA compared to the polystyrene control within the cell experiments. This could be attributed to surface roughness and possible release of residual monomers. Those influences could be investigated in further studies and thus lead to improvement in the additive manufacturing process. In addition, further research focused on the effect of PLA on bone growth should follow. In summary, PLA processed in Fused Deposition Modelling seems to be an attractive material and method for reconstructive surgery because of their biocompatibility and the possibility to produce individually shaped scaffolds. [ABSTRACT FROM AUTHOR]

The quantification of metabolite leakage from damaged mammalian cells to the surrounding medium is of high interest for the processing of samples for metabolomic analysis. It is also of relevance to know the typical time span which is required for a promoted metabolite release through a selectively permeabilized cell membrane. The real-time observation of such a process is difficult since small metabolites cannot be observed directly by optical methods and other more indirect assays can disturb the metabolite concentration itself. However, the diffusion based loss of metabolites from the cytoplasm can be predicted on the basis of reference measurements taken from an easy-to-detect molecule with known diffusion coefficient. In this work, we use green fluorescent protein (GFP) as a marker and model its release from damaged cells using the finite-element method. A correlation between the disrupted membrane area fraction, A, the distribution of membrane ruptures and the rate of GFP efflux, k, has been established. k has been determined experimentally for Chinese hamster ovary cells, which have been damaged mechanically by passage through a micronozzle geometry in a microfluidic system. The immediate GFP release downstream of the micronozzles has been observed in real-time and the corresponding membrane damage has been predicted. On this basis, we calculated the expected times required for the drainage of freely diffusable cytosolic glucose and found a loss of ≈90% within 1 s for a disrupted membrane area fraction of ≈5%. Hence, even minimal membrane damage would lead to a rapid loss of cytosolic metabolites by diffusion unless membrane resealing processes take place. [ABSTRACT FROM AUTHOR]

At Physikalisch-Technische Bundesanstalt, the National Metrology Institute of Germany, a new type of deep ultraviolet scatterometer has been developed and set up. The concept of the system is very variable and versatile, so that many different types of measurements, e.g., classical scatterometry, ellipsometric scatterometry, polarization-dependent reflectometry, and ellipsometry can be performed. The main application is the characterization of linewidth/critical dimension (CD), grating period (pitch), and edge profile of periodically nanostructured surfaces mainly, but not only, on photomasks. Different operation wavelength between 840 and 193 nm can be used, giving also access to a variety of different at-wavelength metrology connected with state-of-the-art photolithography. It allows to adapt and to vary the measurand and measurement geometry to optimize the sensitivity and the unambiguity for the measurement problem. In this paper the concept, design, and performance of the system is described in detail. First measurement examples are shown and current and future applications are discussed. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to identify anatomical areas where resections of oral squamous cell carcinomas (OSCC) are significantly associated with close or positive margins.Materials and methods: This retrospective study included 330 patients with a primary OSCC from 2010 to 2015. Patient and tumour data were categorised into three groups by R-status (R0 [clear], ≥ 5 mm, 185 patients [56.06%]; R1 [positive], < 1 mm, 24 patients [7.27%]; and R0 [close], 1-5 mm, 121 patients [36.67%]).Results: Areas where resections were significantly associated with close or positive margins were the hard palate (p < 0.001), buccal mucosa (p = 0.03), floor of the mouth (p = 0.004), lower alveolar ridge (p = 0.01), retromolar triangle (p = 0.005), and dorsal tongue (p = 0.02).Conclusions: Anatomical areas were identified in the oral cavity where it is challenging to resect OSCCs with an adequate safety margin.Clinical relevance: These results may enable surgeons to achieve a postulated safe distance during tumour resection, leading to a survival benefit for patients. [ABSTRACT FROM AUTHOR]

Background: Scatterometry is a fast, indirect, and nondestructive optical method for quality control in the production of lithography masks. To solve the inverse problem in compliance with the upcoming need for improved accuracy, a computationally expensive forward model that maps geometry parameters to diffracted light intensities has to be defined. Aim: To quantify the uncertainties in the reconstruction of the geometry parameters, a fast-to-evaluate surrogate for the forward model has to be introduced. Approach: We use a nonintrusive polynomial chaos-based approximation of the forward model, which increases speed and thus enables the exploration of the posterior through direct Bayesian inference. In addition, this surrogate allows for a global sensitivity analysis at no additional computational overhead. Results: This approach yields information about the complete distribution of the geometry parameters of a silicon line grating, which in return allows for quantifying the reconstruction uncertainties in the form of means, variances, and higher order moments of the parameters. Conclusions: The use of a polynomial chaos surrogate allows for quantifying both parameter influences and reconstruction uncertainties. This approach is easy to use since no adaptation of the expensive forward model is required. [ABSTRACT FROM AUTHOR]

The following note was inadvertently omitted from the published paper: This work was performed in (partial) fulfillment of the requirements for the first author's obtaining the degree Dr. med. dent. [ABSTRACT FROM AUTHOR]