Quality control in the Netherlands; todays practices and starting points for guidance and future research.

OBJECTIVES: Adequate analytical quality of reported results is primarily ensured by performing internal quality control (iQC). Currently, several different iQC practices are in use. As a prelude to the revision of a Dutch guidance document on analytical QC, a questionnaire was sent out to gain insights in the applied practices and the need for guidance. METHODS: A questionnaire, containing 20 multiple-choice questions with possibilities for explanation and comment on iQC practices and aspects was distributed to all clinical chemistry laboratories within the Netherlands. Results were reported descriptively. RESULTS: Responses were received from 27 clinical laboratories (response 43 %). In 30 % the iQC was based on the analytical characteristics only, while 30 % used a 6-Sigma method, 19 % risk-based beyond 6-Sigma and 22 % used an alternative approach. 89 % of laboratories used a virtual analyzer model for iQC setup within one or more laboratory sites. Practices for determining standard deviation (SD) values included determining SD for each new iQC material (35 %), using historical SD values for new materials (35 %), and incorporating clinical tolerances into the SD value (31 %). Furthermore, 44 % of laboratories used patient moving averages for one or more tests. Daily iQC management was based on either "traffic lights" indicating in or out of control status, and review of all QC charts, often using multiple software systems. CONCLUSIONS: A large heterogeneity of iQC practices in clinical laboratories was observed in the Netherlands. Several starting points for further research and/or guidance were identified, particularly in relation to the determination of SD values, the virtual analyzer model and methods to ensure analyzer equivalence.

Automated calculations for computing the sample-limited spatial resolution in (scanning) transmission electron microscopy.

MATLAB scripts were designed to compute the sample-limited spatial resolution in transmission electron microscopy (TEM) and scanning TEM (STEM) as a function of different microscopy parameters including the electron dose eD, sample geometry, and materials parameters. The scripts can be used to select the optimum microscopy modality and optimize the experimental conditions to achieve the best possible resolution considering the limitations set by both the electron optics and the examined sample. The resolution can be computed as function of the objective opening semi-angle α for TEM and detector opening semi-angle ß for STEM. Optional code for computing a range over the sample thickness t or eD are provided as well, whereby the opening angle is optimized for each data point. The spatial resolution depends on the type of material of the nanoscale object (for example, gold or carbon nanoparticles), the type of matrix holding the objects (for example, water or ice), the depth of the nanoscale object inside the matrix, and eD. The optimization is consistent with the typical situation that carbon nanoparticles are best examined with TEM embedded in a thin matrix (t = 0.1 µm), while STEM is better suited for high atomic number objects such as gold nanoparticles in water, irrespective of t. The script also calculates the reduction of beam broadening in thick samples (t > 1 µm) using bright field STEM.

Strategy for optimizing experimental settings for studying low atomic number colloidal assemblies using liquid phase scanning transmission electron microscopy.

Observing processes of nanoscale materials of low atomic number is possible using liquid phase electron microscopy (LP-EM). However, the achievable spatial resolution (d) is limited by radiation damage. Here, we examine a strategy for optimizing LP-EM experiments based on an analytical model and experimental measurements, and develop a method for quantifying image quality at ultra low electron dose De using scanning transmission electron microscopy (STEM). As experimental test case we study the formation of a colloidal binary system containing 30 nm diameter SiO2 nanoparticles (SiONPs), and 100 nm diameter polystyrene microspheres (PMs). We show that annular dark field (DF) STEM is preferred over bright field (BF) STEM for practical reasons. Precise knowledge of the material's density is crucial for the calculations in order to match experimental data. To calculate the detectability of nano-objects in an image, the Rose criterion for single pixels is expanded to a model of the signal to noise ratio obtained for multiple pixels spanning the image of an object. Using optimized settings, it is possible to visualize the radiation-sensitive, hierarchical low-Z binary structures, and identify both components.

Synthesis of 3,4-Dihydro-2H-Pyrroles from Ketones, Aldehydes, and Nitro Alkanes via Hydrogenative Cyclization.

Syntheses of N-heterocyclic compounds that permit a flexible introduction of various substitution patterns by using inexpensive and diversely available starting materials are highly desirable. Easy to handle and reusable catalysts based on earth-abundant metals are especially attractive for these syntheses. We report here on the synthesis of 3,4-dihydro-2H-pyrroles via the hydrogenation and cyclization of nitro ketones. The latter are easily accessible from three components: a ketone, an aldehyde and a nitroalkane. Our reaction has a broad scope and 23 of the 33 products synthesized are compounds which have not yet been reported. The key to the general hydrogenation/cyclization reaction is a highly active, selective and reusable nickel catalyst, which was identified from a library of 24 earth-abundant metal catalysts.

Nanoscale Faceting and Ligand Shell Structure Dominate the Self-Assembly of Nonpolar Nanoparticles into Superlattices.

Self-assembly of nanoscale structures at liquid-solid interfaces occurs in a broad range of industrial processes and is found in various phenomena in nature. Conventional theory assumes spherical particles and homogeneous surfaces, but that model is oversimplified, and nanoscale in situ observations are needed for a more complete understanding. Liquid-phase scanning transmission electron microscopy (LP-STEM) is used to examine the interactions that direct the self-assembly of superlattices formed by gold nanoparticles (AuNPs) in nonpolar liquids. Varying the molecular coating of the substrate modulates short-range attraction and leads to switching between a range of different geometric structures, including hexagonal close-packed (hcp), simple hexagonal (sh), dodecahedral quasi-crystal (dqc), and body-centered cubic (bcc) lattices, as well as random distributions. Langevin dynamics simulations explain the experimental results in terms of the interplay between nanoparticle faceting, ligand shell structure, and substrate-NP interactions.

Quantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy.

Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.

High temporal-resolution scanning transmission electron microscopy using sparse-serpentine scan pathways.

Scanning transmission electron microscopy (STEM) provides structural analysis with sub-angstrom resolution. But the pixel-by-pixel scanning process is a limiting factor in acquiring high-speed data. Different strategies have been implemented to increase scanning speeds while at the same time minimizing beam damage via optimizing the scanning strategy. Here, we achieve the highest possible scanning speed by eliminating the image acquisition dead time induced by the beam flyback time combined with reducing the amount of scanning pixels via sparse imaging. A calibration procedure was developed to compensate for the hysteresis of the magnetic scan coils. A combination of sparse and serpentine scanning routines was tested for a crystalline thin film, gold nanoparticles, and in an in-situ liquid phase STEM experiment. Frame rates of 92, 23 and 5.8 s-1 were achieved for images of a width of 128, 256, and 512 pixels, respectively. The methods described here can be applied to single-particle tracking and analysis of radiation sensitive materials.

The influence of chromatic aberration on the dose-limited spatial resolution of transmission electron microscopy.

The effect of chromatic aberration (CC) on the spatial resolution in transmission electron microscopy (TEM) was studied in thick specimens in which the sample becomes the limiting factor in the resolution. The sample influences the energy spread of the electron beam, allows only a limited electron dose, and modulates electron scattering events. The experimental set-up consisted of a thin silicon nitride membrane and a silicon wedge containing gold nanoparticles. The resolution was measured as a function of electron dose and sample thickness for different sample configurations and for different microscopy modalities including regular TEM, energy filtered TEM (EFTEM) and CC-corrected TEM. Comparison with an analytical model aided the understanding of the experimental data applied over varied conditions. The general trend for all microscopy modalities was a transition from a noise-limited resolution at low electron dose to a CC-limited resolution at high-dose in the absence of beam blurring. EFTEM required an accurate energy slit offset and an optimal energy spread to energy-slit width ratio to surpass regular TEM. The key advantage of CC correction appeared to be the best possible resolution for larger sample thickness at low electron dose outperforming EFTEM by about fifty percent. Several hypothetical sample configurations relevant to liquid phase electron microscopy were evaluated as well to demonstrate the capabilities of the analytical model and to determine the most optimal microscopy modality for this type of experiment. The analytical model included an automated optimization of the EFTEM settings and may aid in optimizing the sample-limited resolution for experimental analysis and planning.

Verification of water presence in graphene liquid cells.

Graphene liquid cells (GLCs) present the thinnest possible sample enclosures for liquid phase electron microscopy. However, the actual presence of liquid within a GLC is not always guaranteed. Of key importance is to reliably test the presence of the liquid, which is most frequently water or saline. Here, the commonly used methods for verifying the presence of water were evaluated. It is shown that depending on the type of sample, applying a single criterion does not always conclusively verify the presence of water. Testing liquid filling for a specific GLC sample preparation protocol should thus be considered critically. The most reliable method is direct observation of the water exciton peak using electron energy loss spectroscopy (EELS). But if this method cannot be carried out, water filling of the GLC can be verified from a combination of higher contrast in the image, the presence of bubbles, and an oxygen signal in the EEL spectrum, which can be accomplished at a high electron dose in spot mode. Nanoparticle movement does not always occur in a GLC.

Environmental Liquid Cell Technique for Improved Electron Microscopic Imaging of Soft Matter in Solution.

Liquid-phase transmission electron microscopy is a technique for simultaneous imaging of the structure and dynamics of specimens in a liquid environment. The conventional sample geometry consists of a liquid layer tightly sandwiched between two Si3N4 windows with a nominal spacing on the order of 0.5 µm. We describe a variation of the conventional approach, wherein the Si3N4 windows are separated by a 10-µm-thick spacer, thus providing room for gas flow inside the liquid specimen enclosure. Adjusting the pressure and flow speed of humid air inside this environmental liquid cell (ELC) creates a stable liquid layer of controllable thickness on the bottom window, thus facilitating high-resolution observations of low mass-thickness contrast objects at low electron doses. We demonstrate controllable liquid thicknesses in the range 160 ± 34 to 340 ± 71 nm resulting in corresponding edge resolutions of 0.8 ± 0.06 to 1.7 ± 0.8 nm as measured for immersed gold nanoparticles. Liquid layer thickness 40 ± 8 nm allowed imaging of low-contrast polystyrene particles. Hydration effects in the ELC have been studied using poly-N-isopropylacrylamide nanogels with a silica core. Therefore, ELC can be a suitable tool for in situ investigations of liquid specimens.

EGFR Expression in HER2-Driven Breast Cancer Cells.

The epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individual cells allowed the determination of individual receptors label distributions. QD-labeled EGFR was observed with a surface density of (0.5-5) × 101 QDs/µm2, whereas labeled HER2 expression was higher ranging from (2-10) × 102 QDs/µm2. Although most SKBR3 cells expressed low levels of EGFR, an enrichment was observed at large plasma membrane protrusions, and amongst a newly discovered cellular subpopulation termed EGFR-enriched cells.

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy.

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin.

Epidermal growth factor receptor 2 (ErbB2) is found overexpressed in several cancers, such as gastric, and breast cancer, and is, therefore, an important therapeutic target. ErbB2 plays a central role in cancer cell invasiveness, and is associated with cytoskeletal reorganization. In order to study the spatial correlation of single ErbB2 proteins and actin filaments, we applied correlative fluorescence microscopy (FM), and scanning transmission electron microscopy (STEM) to image specifically labeled SKBR3 breast cancer cells. The breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and labeled with quantum dot (QD) nanoparticles attached to specific anti-ErbB2 Affibodies. FM was performed to identify cellular regions with spatially correlated actin and ErbB2 expression. For STEM of the intact plasma membrane of whole cells, the cells were fixed and covered with graphene. Spatial distribution patterns of ErbB2 in the actin rich ruffled membrane regions were examined, and compared to adjacent actin-low regions of the same cell, revealing an association of putative signaling active ErbB2 homodimers with actin-rich regions. ErbB2 homodimers were found absent from actin-low membrane regions, as well as after treatment of cells with Cytochalasin D, which breaks up larger actin filaments. In both latter data sets, a significant inter-label distance of 36 nm was identified, possibly indicating an indirect attachment to helical actin filaments via the formation of heterodimers of ErbB2 with epidermal growth factor receptor (EGFR). The possible attachment to actin filaments was further explored by identifying linear QD-chains in actin-rich regions, which also showed an inter-label distance of 36 nm.

Anti-correlation of HER2 and focal adhesion complexes in the plasma membrane.

Excess presence of the human epidermal growth factor receptor 2 (HER2) as well as of the focal adhesion protein complexes are associated with increased proliferation, migratory, and invasive behavior of cancer cells. A cross-regulation between HER2 and integrin signaling pathways has been found, but the exact mechanism remains elusive. Here, we investigated whether HER2 colocalizes with focal adhesion complexes on breast cancer cells overexpressing HER2. For this purpose, vinculin or talin green fluorescent protein (GFP) fusion proteins, both key constituents of focal adhesions, were expressed in breast cancer cells. HER2 was either extracellularly or intracellularly labeled with fluorescent quantum dots nanoparticles (QDs). The cell-substrate interface was analyzed at the location of the focal adhesions by means of total internal reflection fluorescent microscopy or correlative fluorescence- and scanning transmission electron microscopy. Expression of HER2 at the cell-substrate interface was only observed upon intracellular labeling, and was heterogeneous with both HER2-enriched and -low regions. In contrast to an expected enrichment of HER2 at focal adhesions, an anti-correlated expression pattern was observed for talin and HER2. Our findings suggest a spatial anti-correlation between HER2 and focal adhesion complexes for adherent cells.

Liquid-Phase Electron Microscopy for Soft Matter Science and Biology.

Innovations in liquid-phase electron microscopy (LP-EM) have made it possible to perform experiments at the optimized conditions needed to examine soft matter. The main obstacle is conducting experiments in such a way that electron beam radiation can be used to obtain answers for scientific questions without changing the structure and (bio)chemical processes in the sample due to the influence of the radiation. By overcoming these experimental difficulties at least partially, LP-EM has evolved into a new microscopy method with nanometer spatial resolution and sub-second temporal resolution for analysis of soft matter in materials science and biology. Both experimental design and applications of LP-EM for soft matter materials science and biological research are reviewed, and a perspective of possible future directions is given.

Detecting single ORAI1 proteins within the plasma membrane reveals higher-order channel complexes.

ORAI1 proteins form highly selective Ca2+ channels in the plasma membrane. Crystallographic data point towards a hexameric stoichiometry of ORAI1 channels, whereas optical methods postulated ORAI1 channels to reside as dimers at rest, and other data suggests that they have a tetrameric configuration. Here, liquid-phase scanning transmission electron microscopy (STEM) and quantum dot (QD) labeling was utilized to study the conformation of ORAI1 proteins at rest. To address the question of whether ORAI1 was present as a dimer, experiments were designed using single ORAI1 monomers and covalently linked ORAI1 dimers with either one or two label-binding positions. The microscopic data was statistically analyzed via the pair correlation function. Label pairs were found in all cases, even for concatenated dimers with one label-binding position, which is only possible if a significant fraction of ORAI1 was assembled in larger order oligomers than dimers, binding at least two QDs. This interpretation of the data was consistent with Blue Native PAGE analysis showing that ORAI1 is mainly present as a complex of an apparent molecular mass larger than that calculated for a dimer.

Visualisation of HER2 homodimers in single cells from HER2 overexpressing primary formalin fixed paraffin embedded tumour tissue.

BACKGROUND: HER2 is considered as one of the most important, predictive biomarkers in oncology. The diagnosis of HER2 positive cancer types such as breast- and gastric cancer is usually based on immunohistochemical HER2 staining of tumour tissue. However, the current immunohistochemical methods do not provide localized information about HER2's functional state. In order to generate signals leading to cell growth and proliferation, the receptor spontaneously forms homodimers, a process that can differ between individual cancer cells. MATERIALS AND METHODS: HER2 overexpressing tumour cells were dissociated from formalin-fixed paraffin-embedded (FFPE) patient's biopsy sections, subjected to a heat-induced antigen retrieval procedure, and immobilized on microchips. HER2 was specifically labelled via a two-step protocol involving the incubation with an Affibody-biotin compound followed by the binding of a streptavidin coated quantum dot (QD) nanoparticle. Cells with membrane bound HER2 were identified using fluorescence microscopy, coated with graphene to preserve their hydrated state, and subsequently examined by scanning transmission electron microscopy (STEM) to obtain the locations at the single molecule level. Label position data was statistically analysed via the pair correlation function, yielding information about the presence of HER2 homodimers. RESULTS: Tumour cells from two biopsies, scored HER2 3+, and a HER2 negative control sample were examined. The specific labelling protocol was first tested for a sectioned tissue sample of HER2-overexpressing tumour. Subsequently, a protocol was optimized to study HER2 homodimerization in single cells dissociated from the tissue section. Electron microscopy data showed membrane bound HER2 in average densities of 201-689 proteins/µm2. An automated, statistical analysis of well over 200,000 of measured protein positions revealed the presence of HER2 homodimers in 33 and 55% of the analysed images for patient 1 and 2, respectively. CONCLUSIONS: We introduced an electron microscopy method capable of measuring the positions of individually labelled HER2 proteins in patient tumour cells from which information about the functional status of the receptor was derived. This method could take HER2 testing a step further by examining HER2 homodimerization directly out of tumour tissue and may become important for adjusting a personalized antibody-based drug therapy.

Liquid-Phase Electron Microscopy with Controllable Liquid Thickness.

Liquid-phase electron microscopy (LPEM) is capable of imaging nanostructures and processes in a liquid environment. The spatial resolution achieved with LPEM critically depends on the thickness of the liquid layer surrounding the object of interest. An excessively thick liquid results in broadening of the electron beam and a high background signal that decreases the resolution and contrast of the object in an image. The liquid thickness in a standard liquid cell, consisting of two liquid enclosing membranes separated by spacers, is mainly defined by the deformation of the SiN membrane windows toward the vacuum side, and the effective thickness may differ from the spacer height. Here, we present a method involving a pressure controller setup to balance the pressure difference over the membrane windows, thus manipulating the shape profiles of the used silicon nitride membrane windows. Electron energy loss spectroscopy (EELS) measurements to determine the liquid thickness showed that it is possible to control the thickness precisely during an LPEM experiment by regulating the interior pressure of the liquid cell. We demonstrated atomic resolution on gold nanoparticles and the phase contrast using silica nanoparticles in liquid with controlled thickness.

Dynamics of gold nanoparticle clusters observed with liquid-phase electron microscopy.

The dynamics of processes of nanoparticles such as diffusion, attraction and repulsion, and self-assembly of structures of nanoparticles at the solid-liquid interfaces differ significantly from those occurring for bulk conditions and their fundamental physical rules are still unknown. Here, we used liquid phase scanning transmission electron microscopy (LP-STEM) to study several aspects of nanoparticle dynamics of colloidal chitosan coated gold nanoparticle (TCHIT-AuNP) clusters in a liquid layer enclosed between two SiN membranes. We found that upon beam irradiation using an electron flux of 0.9 e-/sÅ2, the AuNPs assembled in clusters that shifted and rotated with time. The newly formed clusters could join and form larger clusters via a mechanism of oriented attachment. By increasing the electron flux to 6.2 e-/sÅ2, we observed the fragmentation of some of the clusters and TCHIT-AuNPs were exchanged between clusters. At the highest electron flux studied 25 e-/sÅ2, we observed AuNPs moving at a very slow speed compared to Brownian motion in liquid even though they were not directly attached or pinned to the liquid-enclosing membrane. Experiments using branched polyethylenimine (BPEI) coated AuNPs were carried out for comparison.