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.

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.

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.

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.

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.

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.

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.

Reduced Radiation Damage in Transmission Electron Microscopy of Proteins in Graphene Liquid Cells.

Liquid-phase electron microscopy (LPEM) is capable of imaging native (unstained) protein structure in liquid, but the achievable spatial resolution is limited by radiation damage. This damaging effect is more pronounced when targeting small molecular features than for larger structures. The matter is even more complicated because the critical dose that a sample can endure before radiation damage not only varies between proteins but also critically depends on the experimental conditions. Here, we examined the effect of the electron beam on the observed protein structure for optimized conditions using a liquid sample enclosure assembled from graphene sheets. It has been shown that graphene can reduce the damaging effect of electrons on biological materials. We used radiation sensitive microtubule proteins and investigated the radiation damage on these structures as a function of the spatial frequencies of the observed features with transmission electron microscopy (TEM). Microtubule samples were also examined using cryo-electron microscopy (cryo-TEM) for comparison. We used an electron flux of 11 ± 1-16 ± 1 e-/Å2s and obtained a series of images from the same sample region. Our results show that graphene-encapsulated microtubules can maintain their structural features of spatial frequencies of up to 0.20 nm-1 (5 nm), reflecting protofilaments for electron densities of up to 7.2 ± 1.4 × 102 e-/Å2, an order of magnitude higher than measured for frozen microtubules in amorphous ice.

Strategies for Preparing Graphene Liquid Cells for Transmission Electron Microscopy.

A graphene liquid cell for transmission electron microscopy (TEM) uses one or two graphene sheets to separate the liquid from the vacuum in the microscope. In principle, graphene is an excellent material for such an application because it allows the highest possible spatial resolution, provides a flexible covering foil, and effectively protects the liquid from evaporating. Examples in open literature have demonstrated atomic-resolution TEM using small liquid pockets and the coverage of whole biological cells with graphene sheets. A total of three different basic types of liquid cells are discerned: (i) one graphene sheet is used to cover a liquid sample supported by a thin membrane of another material (for example, silicon nitride, SiN), (ii) two graphene sheets pressed together leaving liquid pockets with graphene at both sides, and (iii) a spacer material with liquid pockets covered at both sides by graphene. A total of four different process flows are available for liquid cell assembly, but there is not yet a consensus on the best routes, and a number of variations exist. The key step is the transfer of graphene to a liquid sample, which is complicated by practical issues that arise from imperfections in the graphene sheets, such as cracks. This review provides an overview of these different approaches to assembling graphene liquid cells and discusses the main obstacles and ideas to overcome them with the prospect of developing the nanoscale technology needed for graphene liquid cells so that they become available on a routine basis for electron microscopy in liquid. It also provides guidance in selecting the appropriate type of graphene liquid cell and the best assembly method for a specific experiment.

Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy.

The spatial distribution of the human epidermal growth factor 2 (HER2) receptor in the plasma membrane of SKBR3 and HCC1954 breast cancer cells was studied. The receptor was labeled with quantum dot nanoparticles, and fixed whole cells were imaged in their native liquid state with environmental scanning electron microscopy using scanning transmission electron microscopy detection. The locations of individual HER2 positions were determined in a total plasma membrane area of 991 µm2 for several SKBR3 cells and 1062 µm2 for HCC1954 cells. Some of the HER2 receptors were arranged in a linear chain with interlabel distances of 40 ± 7 and 32 ± 10 nm in SKBR3 and HCC1954 cells, respectively. The finding was tested against randomly occurring linear chains of six or more positions, from which it was concluded that the experimental finding is significant and did not arise from random label distributions. Because the measured interlabel distance in the HER2 chains is similar to the 36-nm helix-repetition distance of actin filaments, it is proposed that a linking mechanism between HER2 and actin filaments leads to linearly aligned oligomers.

The 2018 correlative microscopy techniques roadmap.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.

The Influence of Beam Broadening on the Spatial Resolution of Annular Dark Field Scanning Transmission Electron Microscopy.

The spatial resolution of aberration-corrected annular dark field scanning transmission electron microscopy was studied as function of the vertical position z within a sample. The samples consisted of gold nanoparticles (AuNPs) positioned in different horizontal layers within aluminum matrices of 0.6 and 1.0 µm thickness. The highest resolution was achieved in the top layer, whereas the resolution was reduced by beam broadening for AuNPs deeper in the sample. To examine the influence of the beam broadening, the intensity profiles of line scans over nanoparticles at a certain vertical location were analyzed. The experimental data were compared with Monte Carlo simulations that accurately matched the data. The spatial resolution was also calculated using three different theoretical models of the beam blurring as function of the vertical position within the sample. One model considered beam blurring to occur as a single scattering event but was found to be inaccurate for larger depths of the AuNPs in the sample. Two models were adapted and evaluated that include estimates for multiple scattering, and these described the data with sufficient accuracy to be able to predict the resolution. The beam broadening depended on z 1.5 in all three models.

The stoichiometry of the TMEM16A ion channel determined in intact plasma membranes of COS-7 cells using liquid-phase electron microscopy.

TMEM16A is a membrane protein forming a calcium-activated chloride channel. A homodimeric stoichiometry of the TMEM16 family of proteins has been reported but an important question is whether the protein resides always in a dimeric configuration in the plasma membrane or whether monomers of the protein are also present in its native state within in the intact plasma membrane. We have determined the stoichiometry of the human (h)TMEM16A within whole COS-7 cells in liquid. For the purpose of detecting TMEM16A subunits, single proteins were tagged by the streptavidin-binding peptide within extracellular loops accessible by streptavidin coated quantum dot (QD) nanoparticles. The labeled proteins were then imaged using correlative light microscopy and environmental scanning electron microscopy (ESEM) using scanning transmission electron microscopy (STEM) detection. The locations of 19,583 individual proteins were determined of which a statistical analysis using the pair correlation function revealed the presence of a dimeric conformation of the protein. The amounts of detected label pairs and single labels were compared between experiments in which the TMEM16A SBP-tag position was varied, and experiments in which tagged and non-tagged TMEM16A proteins were present. It followed that hTMEM16A resides in the plasma membrane as dimer only and is not present as monomer. This strategy may help to elucidate the stoichiometry of other membrane protein species within the context of the intact plasma membrane in future.

Anisotropic Shape Changes of Silica Nanoparticles Induced in Liquid with Scanning Transmission Electron Microscopy.

Liquid-phase transmission electron microscopy (TEM) is used for in-situ imaging of nanoscale processes taking place in liquid, such as the evolution of nanoparticles during synthesis or structural changes of nanomaterials in liquid environment. Here, it is shown that the focused electron beam of scanning TEM (STEM) brings about the dissolution of silica nanoparticles in water by a gradual reduction of their sizes, and that silica redeposites at the sides of the nanoparticles in the scanning direction of the electron beam, such that elongated nanoparticles are formed. Nanoparticles with an elongation in a different direction are obtained simply by changing the scan direction. Material is expelled from the center of the nanoparticles at higher electron dose, leading to the formation of doughnut-shaped objects. Nanoparticles assembled in an aggregate gradually fuse, and the electron beam exposed section of the aggregate reduces in size and is elongated. Under TEM conditions with a stationary electron beam, the nanoparticles dissolve but do not elongate. The observed phenomena are important to consider when conducting liquid-phase STEM experiments on silica-based materials and may find future application for controlled anisotropic manipulation of the size and the shape of nanoparticles in liquid.

The Effect of Electron Beam Irradiation in Environmental Scanning Transmission Electron Microscopy of Whole Cells in Liquid.

Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e-/Å2 toward 103 e-/Å2. This dose range was sufficient to determine the EGFR locations with nanometer resolution and to distinguish between monomers and dimers. Various different forms of radiation damage became visible at higher doses, including severe dislocation, and the dissolution of labels.

Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy.

ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the pair correlation function and, in addition, an analysis of cluster size and frequency was performed. ORAI1 was found to be present in hexamers in a small fraction only, and ORAI1 resided mostly in monomers and dimers.

Writing silica structures in liquid with scanning transmission electron microscopy.

Silica nanoparticles are imaged in solution with scanning transmission electron microscopy (STEM) using a liquid cell with silicon nitride (SiN) membrane windows. The STEM images reveal that silica structures are deposited in well-defined patches on the upper SiN membranes upon electron beam irradiation. The thickness of the deposits is linear with the applied electron dose. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrate that the deposited patches are a result of the merging of the original 20 nm-diameter nanoparticles, and that the related surface roughness depends on the electron dose rate used. Using this approach, sub-micrometer scale structures are written on the SiN in liquid by controlling the electron exposure as function of the lateral position.

Exceptionally Slow Movement of Gold Nanoparticles at a Solid/Liquid Interface Investigated by Scanning Transmission Electron Microscopy.

Gold nanoparticles were observed to move at a liquid/solid interface 3 orders of magnitude slower than expected for the movement in a bulk liquid by Brownian motion. The nanoscale movement was studied with scanning transmission electron microscopy (STEM) using a liquid enclosure consisting of microchips with silicon nitride windows. The experiments involved a variation of the electron dose, the coating of the nanoparticles, the surface charge of the enclosing membrane, the viscosity, and the liquid thickness. The observed slow movement was not a result of hydrodynamic hindrance near a wall but instead explained by the presence of a layer of ordered liquid exhibiting a viscosity 5 orders of magnitude larger than a bulk liquid. The increased viscosity presumably led to a dramatic slowdown of the movement. The layer was formed as a result of the surface charge of the silicon nitride windows. The exceptionally slow motion is a crucial aspect of electron microscopy of specimens in liquid, enabling a direct observation of the movement and agglomeration of nanoscale objects in liquid.

Matched Backprojection Operator for Combined Scanning Transmission Electron Microscopy Tilt- and Focal Series.

Combined tilt- and focal series scanning transmission electron microscopy is a recently developed method to obtain nanoscale three-dimensional (3D) information of thin specimens. In this study, we formulate the forward projection in this acquisition scheme as a linear operator and prove that it is a generalization of the Ray transform for parallel illumination. We analytically derive the corresponding backprojection operator as the adjoint of the forward projection. We further demonstrate that the matched backprojection operator drastically improves the convergence rate of iterative 3D reconstruction compared to the case where a backprojection based on heuristic weighting is used. In addition, we show that the 3D reconstruction is of better quality.