In-focus electron microscopy of frozen-hydrated biological samples with a Boersch phase plate

We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C_s corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of <0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.     

Linking the proteins--elucidation of proteome-scale networks using mass spectrometry

Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks.     

Cellular analysis by open-source software for affordable cytometry

Image cytometry is an important technique in affordable healthcare and cellular research. Some efforts toward establishing a personal, low-cost cytometer have been described in the literature. However, a self-assembled fluorescence microscope requires software for cytometric analysis. There are some open-source image-based software analysis applications available. However, for a quantitative analysis of images, software that can generate data comparable to those of previously evaluated cytometric analyses programs is required. Hence, the aim of this study is to compare results of a commercially available image cytometry program to data obtained using the open-source software CellProfiler (CP). Leukocytes and fluorescent bead images obtained using a Laser Scanning Cytometer were analyzed by CP and the results compared with those of conventional cytometric analyses' programs. Algorithms were developed enabling the analysis of leukocytes and beads by CP. CP provided similar results to those obtained by the cytometer software. Hallmark parameters, including cell count and fluorescence intensity, revealed a high correlation in the analysis of both programs. Therefore, CP is appropriate for cellular analysis on a self-assembled microscope, thereby enabling affordable cytometry.     

A method to determine the local surface profile from reconstructed exit waves

Reconstructed exit waves are useful to quantify unknown structure parameters such as the position and composition of the atom columns at atomic scale. Existing techniques provide a complex wave in a flat plane which is close to the plane where the electrons leave the atom columns. However, due to local deviation in the flatness of the exit surface, there will be an offset between the plane of reconstruction and the actual exit of a specific atom column. Using the channelling theory, it has been shown that this defocus offset can in principle be determined atom column-by-atom column. As such, the surface roughness could be quantified at atomic scale. However, the outcome strongly depends on the initial plane of reconstruction especially in a crystalline structure. If this plane is further away from the true exit, the waves of the atom columns become delocalized and interfere mutually which strongly complicates the interpretation of the exit wave in terms of the local structure. In this paper, we will study the delocalization with defocus using the channelling theory in a more systematic way.     

Young's modulus repeatability assessment using cycling compression loading on cancellous bone

For various applications, precision of the Young's modulus of cancellous bone specimens is needed. However, measurement variability is rarely given. The aim of this study was to assess the Young's modulus repeatability using a uniaxial cyclic compression protocol on embedded specimens of human cancellous bone. Twelve specimens from 12 human calcanei were considered. The specimens were first defatted and then 1 or 2 mm at the ends were embedded in an epoxy resin. The compression experiment consists in applying 20 compressive cycles between 0.2 per cent and 0.6 per cent strain with a 2 Hz loading frequency. The coefficient of variation of the current protocol was found to be 1.2 percent. This protocol showed variability similar to the end-cap technique (considered as a reference). It can be applied on porous specimen (especially human bone) and requires minimal bone length to limit end-artifact variability. The current method could be applied in association with noninvasive measurements (such as ultrasound) with full compatibility. This possibility opens the way for bone damage follow-up based on Young's modulus monitoring.     

Pressure waves in the aorta during isolated abdominal belt loading: the magnitude, phasing, and attenuation

While rupture of the aorta is a leading cause of sudden death following motor vehicle crashes, the specific mechanism that causes this injury is not currently well understood. Aortic ruptures occurring in the field are likely due to a complex combination of contributing factors such as acceleration, compression of the chest, and increased pressure within the aorta. The objective of the current study was to investigate one of these factors in more detail than has been done previously; specifically, to investigate the in situ intra-aortic pressure generated during isolated belt loading to the abdomen. Ten juvenile swine were subjected to dynamic belt loads applied to the abdomen. Intraaortic pressure was measured at multiple locations to assess the magnitude and propagation of the resulting blood pressure wave. The greatest average peak pressure (113.6 +/- 43.5 kPa) was measured in the abdominal aorta. Pressures measured in the thoracic aorta and aortic arch were 70 per cent and 50 per cent, respectively, that measured in the abdominal aorta. No macroscopic aortic trauma was observed. To the authors' knowledge the present study is the first one to document the presence, propagation, and attenuation of a transient pressure wave in the aorta generated by abdominal belt loading. The superiorly moving wave is sufficient to generate hydrostatic and intimal shear stress in the aorta, possibly contributing to the hypothesized mechanisms of traumatic aortic rupture.     

Computer-aided methods in bespoke breast prosthesis design and fabrication

This case study presents a method of utilizing computer-aided design technologies to provide bespoke, external breast prostheses. The technique is illustrated through a case study of a mastectomy patient. Photogrammetry methods were used to capture the breast form when supported by a brassiere and the unsupported breast and defect side. Computer-aided design techniques were used to generate a digital prosthesis based upon the supported breast shape and with a fitting surface that matched the defect side. Furthermore, a two-part mould was designed and fabricated using rapid prototyping methods. A colour-matched prosthesis was then fabricated in a gel-based, platinum-cured silicone.     

Elimination and accumulation of polycyclic aromatic hydrocarbons in urban stormwater wet detention ponds

The concentrations of polycyclic aromatic hydrocarbons (PAHS) in water and sediments of seven wet detention ponds receiving urban stormwater were investigated. The ponds comprised traditional wet detention ponds with a permanent wet volume and a storage volume as well as ponds that were expanded with sand filters and other means to improve the removal of micropollutants. The concentrations of sigmaPAH in the sediments varied between 6 +/- 5 and 2,222 +/- 603 ng g(-1) dry weight (mean +/- standard deviation), and were highest in the ponds with lower pond volume per catchment area and did not clearly reflect different activities in the catchments. In general, the concentrations of PAHS in the sediments decreased from inlet to outlet, especially in the systems with good conditions for sedimentation such as systems with flow perpendicular sand dikes and extensive submerged vegetation. High molecular weight PAHs were predominant in the sediments indicating the pyrogenic origin of the PAHS. There was no correlation between PAH species concentrations in water or sediments and their hydrophobicity (log K(ow)). PAH concentrations in water fluctuated in response to intensity and frequency of rain events, whereas concentrations in the sediments integrated the pollutant load over time. Pond systems expanded with sand filters and other technologies to enhance removal of micropollutants consistently had concentrations of PAHS in the effluents below the detection level.     

Flow measurements in sewers based on image analysis: automatic flow velocity algorithm

Discharges of combined sewer overflows (CSOs) and stormwater are recognized as an important source of environmental contamination. However, the harsh sewer environment and particular hydraulic conditions during rain events reduce the reliability of traditional flow measurement probes. An in situ system for sewer water flow monitoring based on video images was evaluated. Algorithms to determine water velocities were developed based on image-processing techniques. The image-based water velocity algorithm identifies surface features and measures their positions with respect to real world coordinates. A web-based user interface and a three-tier system architecture enable remote configuration of the cameras and the image-processing algorithms in order to calculate automatically flow velocity on-line. Results of investigations conducted in a CSO are presented. The system was found to measure reliably water velocities, thereby providing the means to understand particular hydraulic behaviors.     

The application of model predictive control of ammonia nitrogen in an activated sludge process

In this paper a model predictive controller (MPC) for ammonia nitrogen is presented and evaluated in a real activated sludge process. A reduced nonlinear mathematical model based on mass balances is used to model the ammonia nitrogen in the activated sludge plant. An MPC algorithm that minimises only the control error at the end of the prediction interval is applied. The results of the ammonia MPC were compared with the results of the ammonia feedforward-PI and ammonia PI controllers from our previous study. The ammonia MPC and ammonia feedforward-PI controller give better results in terms of ammonia removal and aeration energy consumption than the ammonia PI controller because of the measurable disturbances used. On the other hand, with the ammonia MPC, comparable or even slightly poorer results than with the ammonia feedforward-PI controller are obtained. Further improvements to the MPC could be possible with an improved accuracy of the nonlinear reduced model of the ammonia nitrogen, more sophisticated control criteria used inside the controller and the extension of the problem from univariable ammonia to multivariable total nitrogen control.