TY - JOUR A1 - Rühle, Bastian A1 - Hodoroaba, Vasile-Dan T1 - Towards Automated Electron Microscopy Image Segmentation for Nanoparticles of Complex Shape by Convolutional Neural Networks N2 - In this contribution different ways are explored with the aim to generate suitable training data for ‘non-ideal’ samples using various approaches, e.g., computer-generated images or unsupervised learning algorithms such as generative adversarial networks (GANs). We used these data to train simple CNNs to produce segmentation masks of SEM images and tested the trained networks on real SEM images of complex nanoparticle samples. The novel use of CNN for the automated analysis of the size of nanoparticles of complex shape and with a high degree of agglomeration has proved to be a promising tool for the evaluation of particle size distribution on a large number of constituent particles. Further development and validation of the preliminary model, respectively larger training and validation data sets are necessary. KW - Nanoparticles KW - Convolutional neural networks KW - Image segmentation KW - Electron microscopy KW - Automatisation PY - 2020 DO - https://doi.org/10.1017/S1431927620017262 VL - 26 IS - S2 SP - 1188 EP - 1189 PB - Cambridge University Press CY - Cambridge, UK AN - OPUS4-51773 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Rühle, Bastian A1 - Krumrey, Julian Frederic A1 - Hodoroaba, Vasile-Dan T1 - Workflow towards automated segmentation of agglomerated, non‑spherical particles from electron microscopy images using artificial neural networks N2 - We present a workflow for obtaining fully trained artificial neural networks that can perform automatic particle segmentations of agglomerated, non-spherical nanoparticles from scanning electron microscopy images “from scratch”, without the need for large training data sets of manually annotated images. The whole process only requires about 15 minutes of hands-on time by a user and can typically be finished within less than 12 hours when training on a single graphics card (GPU). After training, SEM image analysis can be carried out by the artificial neural network within seconds. This is achieved by using unsupervised learning for most of the training dataset generation, making heavy use of generative adversarial networks and especially unpaired image-to-image translation via cycle-consistent adversarial networks. We compare the segmentation masks obtained with our suggested workflow qualitatively and quantitatively to state-of-the-art methods using various metrics. Finally, we used the segmentation masks for automatically extracting particle size distributions from the SEM images of TiO2 particles, which were in excellent agreement with particle size distributions obtained manually but could be obtained in a fraction of the time. KW - Electron microscopy KW - Neural networks KW - Artificial intelligence KW - Image segmentation KW - Automated image analysis PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-522454 DO - https://doi.org/10.1038/s41598-021-84287-6 VL - 11 IS - 1 SP - 4942 PB - Springer Nature AN - OPUS4-52245 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Özelci, Ersan A1 - Rühle, Bastian A1 - Weigert, Florian A1 - Lubotzky, B. A1 - Kewes, G. A1 - Resch-Genger, Ute A1 - Benson, O. T1 - Quantitative measurements of the pH-sensitive quantum yield of fluorophores in mesoporous silica thin films using a drexhage-type experiment N2 - The photoluminescence quantum yield characterizes the performance of emitters for applications in optical devices, as reporters or probes in material and analytical sciences, and for sensing applications. Quantum yield measurements are challenging for luminescent molecules and nanocrystals immobilized in thin films for many sensor applications, particularly if spatially resolved quantitative luminescence information is desired. We show here that a Drexhage-type experiment, where a silver-coated millimeter-sized sphere is used to modify the local density of states, can provide an elegant approach to counter this challenge. As a representative example of the potential of this method, we measure the pH-dependent photoluminescence quantum yield of fluorescein isothiocyanate bound to a thin mesoporous silica film. The results were compared with those of the studies on the pH dependence of the same dye in solution. We found that our approach can link single fluorophore studies to ensemble measurements and pave the way for the spatially resolved fluorescence measurements of ultralow concentrations of emitters utilized as optically active elements and reporters in thin sensor films or incorporated into membranes. KW - Fluorescence KW - Quantum yield KW - Method KW - pH KW - Dye KW - Sensor KW - Fluorescein KW - Film KW - Silica KW - Single molecule KW - Lifetime KW - Absolute quantum yield PY - 2019 DO - https://doi.org/10.1021/acs.jpcc.9b03917 SN - 1932-7447 SN - 1932-7455 VL - 123 IS - 33 SP - 20468 EP - 20475 PB - ACS Publications AN - OPUS4-48984 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Saleh, Maysoon I. A1 - Panas, I. A1 - Frenzel, Florian A1 - Würth, Christian A1 - Rühle, Bastian A1 - Slominskii, Y. A1 - Demchenko, A. A1 - Resch-Genger, Ute T1 - Sensitization of upconverting nanoparticles with a NIR-emissive cyanine dye using a micellar encapsulation approach N2 - Photon upconversion nanomaterials have a wide range of applications, including biosensing and deep-tissue imaging. Their typically very weak and narrow absorption bands together with their size dependent luminescence efficiency can limit their application potential. This has been addressed by increasingly sophisticated core-shell particle architectures including the sensitization with organic dyes that strongly absorb in the near infrared (NIR). In this work, we present a simple water-dispersible micellar system that features energy transfer from the novel NIR excitable dye, 1859 SL with a high molar absorption coefficient and a moderate fluorescence quantum yield to oleate-capped NaYF4:20%Yb(III), 2%Er(III) upconversion nanoparticles (UCNP) upon 808 nm excitation. The micelles were formed using the surfactants Pluronic F-127 and Tween 80 to produce a hydrophilic dye-UCNP system. Successful energy transfer from the dye to the UCNP could be confirmed by emission measurements that revealed the occurrence of upconversion emission upon excitation at 808 nm and an enhancement of the green Er(III) emission compared to direct Er(III) excitation at 808 nm. KW - Upconversion nanoparticles KW - Sensitization KW - NIR dyes KW - Energy transfer PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-472300 DO - https://doi.org/10.1088/2050-6120/aafe1f SN - 2050-6120 VL - 7 IS - 1 (Special issue: Upconversion Methods, Applications and Materials) SP - 014003-1 EP - 014003-9 PB - IOP AN - OPUS4-47230 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Roloff, Alexander A1 - Nirmalananthan-Budau, Nithiya A1 - Rühle, Bastian A1 - Borcherding, H. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Quantification of Aldehydes on Polymeric Microbead Surfaces via Catch and Release of Reporter Chromophores N2 - Aldehyde moieties on 2D-supports or microand nanoparticles can function as anchor groups for the attachment of biomolecules or as reversible binding sites for proteins on cell surfaces. The use of aldehyde-based materials in bioanalytical and medical settings calls for reliable methods to detect and quantify this functionality. We report here on a versatile concept to quantify the accessible aldehyde moieties on particle surfaces through the specific binding and subsequent release of small reporter molecules such as fluorescent dyes and nonfluorescent chromophores utilizing acylhydrazone formation as a reversible covalent labeling strategy. This is representatively demonstrated for a set of polymer microparticles with different aldehyde labeling densities. Excess reporter molecules can be easily removed by washing, eliminating inaccuracies caused by unspecific adsorption to hydrophobic surfaces. Cleavage of hydrazones at acidic pH assisted by a carbonyl trap releases the fluorescent reporters rapidly and quasi-quantitatively and allows for their fluorometric detection at low concentration. Importantly, this strategy separates the signal-generating molecules from the bead surface. This circumvents common issues associated with light scattering and signal distortions that are caused by binding-induced changes in reporter fluorescence as well as quenching dye− dye interactions on crowded particle surfaces. In addition, we demonstrate that the release of a nonfluorescent chromophore via disulfide cleavage and subsequent quantification by absorption spectroscopy gives comparable results, verifying that both assays are capable of rapid and sensitive quantification of aldehydes on microbead surfaces. These strategies enable a quantitative comparison of bead batches with different functionalization densities, and a qualitative prediction of their coupling efficiencies in bioconjugations, as demonstrated in reductive amination reactions with Streptavidin. KW - Fluorescent label KW - Surface group quantification KW - Polymer particle KW - Cleavable linker KW - Catch and release assay PY - 2019 DO - https://doi.org/10.1021/acs.analchem.8b05515 SN - 0003-2700 SN - 1520-6882 VL - 91 IS - 14 SP - 8827 EP - 8834 PB - ACS Publications AN - OPUS4-48284 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Modena, Mario M. A1 - Rühle, Bastian A1 - Burg, Thomas P. A1 - Wuttke, Steffan T1 - Nanoparticle characterization: What to measure? N2 - What to measure? is a key question in nanoscience, and it is not straightforward to address as different physicochemical properties define a nanoparticle sample. Most prominent among these properties are size, shape, surface charge, and porosity. Today researchers have an unprecedented variety of measurement techniques at their disposal to assign precise numerical values to those parameters. However, methods based on different physical principles probe different aspects, not only of the particles themselves, but also of their preparation history and their environment at the time of measurement. Understanding these connections can be of great value for interpreting characterization results and ultimately controlling the nanoparticle structure–function relationship. Here, the current techniques that enable the precise measurement of these fundamental nanoparticle properties are presented and their practical advantages and disadvantages are discussed. Some recommendations of how the physicochemical parameters of nanoparticles should be investigated and how to fully characterize these properties in different environments according to the intended nanoparticle use are proposed. The intention is to improve comparability of nanoparticle properties and performance to ensure the successful transfer of scientific knowledge to industrial real‐world applications. KW - Nanoparticle characterization KW - Nanoparticles KW - Porosity KW - Shape KW - Size PY - 2019 DO - https://doi.org/10.1002/adma.201901556 SN - 0935-9648 SN - 1521-4095 VL - 31 IS - 32 SP - 1901556, 1 EP - 26 PB - Wiley AN - OPUS4-49129 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nirmalananthan-Budau, Nithiya A1 - Rühle, Bastian A1 - Geißler, Daniel A1 - Moser, Marko A1 - Kläber, Christopher A1 - Schäfer, A. A1 - Resch-Genger, Ute T1 - Multimodal cleavable reporters for quantifying carboxy and amino groups on organic and inorganic nanoparticles N2 - Organic and inorganic nanoparticles (NPs) are increasingly used as drug carriers, fluorescent sensors, and multimodal labels in the life and material sciences. These applications require knowledge of the chemical nature, total number of surface groups, and the number of groups accessible for subsequent coupling of e.g., antifouling ligands, targeting bioligands, or sensor molecules. To establish the concept of catch-and-release assays, cleavable probes were rationally designed from a quantitatively cleavable disulfide moiety and the optically detectable reporter 2-thiopyridone (2-TP). For quantifying surface groups on nanomaterials, first, a set of monodisperse carboxy-and amino-functionalized, 100 nm-sized polymer and silica NPs with different surface group densities was synthesized. Subsequently, the accessible functional groups (FGs) were quantified via optical spectroscopy of the cleaved off reporter after its release in solution. Method validation was done with inductively coupled plasma optical emission spectroscopy (ICP-OES) utilizing the sulfur atom of the cleavable probe. This comparison underlined the reliability and versatility of our probes, which can be used for surface group quantification on all types of transparent, scattering, absorbing and/or fluorescent particles. The correlation between the total and accessible number of FGs quantified by conductometric titration, qNMR, and with our cleavable probes, together with the comparison to results of conjugation studies with differently sized biomolecules reveal the potential of catch-and-release reporters for surface analysis. Our findings also underline the importance of quantifying particularly the accessible amount of FGs for many applications of NPs in the life sciences. KW - Advanced Materials KW - Surface Chemistry KW - Organic–inorganic nanostructures KW - Funtional Groups KW - Quantitative Analysis PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-499081 DO - https://doi.org/10.1038/s41598-019-53773-3 VL - 9 SP - 17577-1 EP - 17577-11 PB - Springer Nature CY - London AN - OPUS4-49908 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Mrkwitschka, Paul A1 - Abram, Sarah-Luise A1 - Rühle, Bastian A1 - Hodoroaba, Vasile-Dan T1 - Understanding Correlative Electron Microscopy Imaging with SEM, STEM-in-SEM and TEM for the Accurate Characterization of Size and Shape of Iron Oxide Nanoparticles N2 - The recently certified reference material (CRM) BAM-N012 as cubical iron oxide FeOx nanoparticles (NPs) of 8 nm area equivalent square edge length (ESL) and the RM candidate BAM-N013 as nearly spherical NPs of 22 nm size were analyzed in detail by electron microscopy (EM). For the metrological characterization with SEM, STEM in SEM and TEM, the understanding of the imaging contrasts and sensitivities, and the correct interpretation of the (art)effects which are inherent to each detection mode is necessary. The same sample areas with NPs deposited on a TEM grid were analyzed by two SEM acquisition modes, i. e. SE InLens and STEM in SEM using a dedicated transmission sample holder, and further, correlatively, analyzed with TEM. With increasing kV, SE InLens shows increasing particle size (unless overcharging at the particle boundaries is filtered), as a known effect. For STEM-in-SEM the particle size decreases significantly and individual particles are identified easier (at 2 kV only a few single particles can be detected automatically). ❑ Documentation of the sample preparation and measurement conditions (including optimization process) is important for reproducibility. ❑ Plasma cleaning, analysis in the transmission mode at SEM is recommended for FeOx NPs. ❑ Selection of the threshold algorithm can significantly alter the reported ECD. KW - Nanoparticles KW - Iron oxide KW - Electron microscopy KW - Reference materials KW - Particle size distribution KW - Imaging PY - 2024 DO - https://doi.org/10.1093/mam/ozae044.339 VL - 30 IS - Supplement_1 SP - 710 EP - 711 PB - Oxford University Press (OUP) AN - OPUS4-61007 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Saleh, Maysoon I. A1 - Rühle, Bastian A1 - Wang, Shu A1 - Radnik, Jörg A1 - You, Yi A1 - Resch-Genger, Ute T1 - Assessing the protective effects of different surface coatings on NaYF4:YB3+, Er3+, upconverting nanoparticles in buffer and DMEM N2 - We studied the dissolution behavior of β NaYF4:Yb(20%), Er(2%) UCNP of two different sizes in biologically relevant media i.e., water (neutral pH), phosphate buffered saline (PBS), and Dulbecco’s modified Eagle medium (DMEM) at different temperatures and particle concentrations. Special emphasis was dedicated to assess the influence of different surface functionalizations, particularly the potential of mesoporous and microporous silica shells of different thicknesses for UCNP stabilization and protection. Dissolution was quantified electrochemically using a fluoride ion selective electrode (ISE) and by inductively coupled plasma optical emission spectrometry (ICP OES). In addition, dissolution was monitored fluorometrically. These experiments revealed that a thick microporous silica shell drastically decreased dissolution. Our results also underline the critical influence of the chemical composition of the aqueous environment on UCNP dissolution. In DMEM, we observed the formation of a layer of adsorbed molecules on the UCNP surface that protected the UCNP from dissolution and enhanced their fluorescence. Examination of this layer by X ray photoelectron spectroscopy (XPS) and mass spectrometry (MS) suggested that mainly phenylalanine, lysine, and glucose are adsorbed from DMEM. These findings should be considered in the future for cellular toxicity studies with UCNP and other nanoparticles and the design of new biocompatible surface coatings. KW - Fluorescence KW - Lifetime KW - Method KW - Quantification KW - Stability KW - Coating KW - Surface chemistry KW - Lanthanide KW - Fluoride KW - Electrochemistry KW - ICP-OES KW - Upconversion KW - Nano KW - Particle KW - Aging KW - Quality assurance KW - Mass spectrometry KW - XPS PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-515984 DO - https://doi.org/10.1038/s41598-020-76116-z SN - 2045-2322 VL - 10 IS - 1 SP - 19318-1 EP - 19318-11 PB - Springer Nature CY - London AN - OPUS4-51598 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Nordholt, Niclas A1 - O'Hara, Kate A1 - Resch-Genger, Ute A1 - Blaskovich, M. A1 - Rühle, Bastian A1 - Schreiber, Frank T1 - A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria N2 - Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA‘s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action. KW - Antimicrobial resistance KW - Bacteria KW - Disinfection KW - Biocides PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-563811 DO - https://doi.org/10.3389/fmicb.2022.1023326 SN - 1664-302X IS - 13 SP - 1 EP - 13 PB - Frontiers Media CY - Lausanne AN - OPUS4-56381 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -