Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy

Structure of bacterial biofilms may be investigated using several variants of scanning electron microscopy (SEM). We apply lyophilization to prepare nitrifying bacterial biofilm for conventional SEM imaging in high-vacuum mode (CSEM). We therefore replace standard biofilm fixation in glutaraldehyde cross-linking, ethanol dehydration, and critical-point drying (CPD) with less-invasive low-temperature drying by sublimation in vacuum. We compare this approach with: (1) standard preparation with glutaraldehyde fixation, ethanol dehydration, and CPD before CSEM, (2) cryo-sputter preparation of rapidly frozen biofilm in hydrated state (cryo-SEM), and (3) in situ observation without any sample pretreatment in environmental SEM. Combined imaging with these modalities revealed two distinct immobilization patterns on the polyurethane foam: (1) large irregular aggregates (flocs) of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) biofilm threads adherent to the surface of polyurethane foam. Our results indicate that lyophilization was suitable for preservation of bacterial cells and many forms of structure of extracellular matrix. The lyophilized material could be imaged with high resolution (using CSEM) to generate structural information complementary to that obtained with other SEM techniques.     

Taking back control of privacy: a novel framework for preserving cloud-based firewall policy confidentiality

As the cloud computing paradigm evolves, new types of cloud-based services have become available, including security services. Some of the most important and most commonly adopted security services are firewall services. These cannot be easily deployed in a cloud, however, because of a lack of mechanisms preserving firewall policy confidentiality. Even if they were provided, the customer traffic flowing through the Cloud Service Provider infrastructure would still be exposed to eavesdropping and information gaining by performing analysis. To bypass these issues, the following article introduces a novel framework, known as the Ladon Hybrid Cloud, for preserving cloud-based firewall policy confidentiality. It is shown that in this framework, a high level of privacy is provided thanks to leveraging an anonymized firewall approach and a hybrid cloud model. A number of optimization techniques, which help to further improve the Ladon Hybrid Cloud privacy level, are also introduced. Finally, analysis performed on the framework shows that it is possible to find a trade-off between the Ladon Hybrid Cloud privacy level, its congestion probability, and efficiency. This argument has been demonstrated through the results of conducted experiments.     

Single- and two-photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes

Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single- and two-photon spectral properties of a strong yellowish-green AF confined to the plasma-membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three-dimensional aggregates or as monolayers. The excitation-emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde-fixed cells.     

Application of detector precision characteristics and histogram packing for compression of biological fluorescence micrographs

Modern applications of biological microscopy such as high-content screening (HCS), 4D imaging, and multispectral imaging may involve collection of thousands of images in every experiment making efficient image-compression techniques necessary. Reversible compression algorithms, when used with biological micrographs, provide only a moderate compression ratio, while irreversible techniques obtain better ratios at the cost of removing some information from images and introducing artifacts. We construct a model of noise, which is a function of signal in the imaging system. In the next step insignificant intensity levels are discarded using intensity binning. The resultant images, characterized by sparse intensity histograms, are coded reversibly. We evaluate compression efficiency of combined reversible coding and intensity depth-reduction using single-channel 12-bit light micrographs of several subcellular structures. We apply local and global measures of intensity distribution to estimate maximum distortions introduced by the proposed algorithm. We demonstrate that the algorithm provides efficient compression and does not introduce significant changes to biological micrographs. The algorithm preserves information content of these images and therefore offers better fidelity than standard irreversible compression method JPEG2000.     

AERODYNAMIC ATOMIZATION OF POLYMERIC SOLUTIONS

A helium activated firing device was used to propel viscoelastic fluid slugs (350 cc) to Mach I velocities. Aerodynamic forces disrupt the slug which eventually transforms into a cloud of droplets that fall on the ground below. Various concentrations and types of polymethyl methacrylate (PMM A) in diethylmalonate (DEM) were disseminated. The resultant average drop size for each trial was measured and shown to significantly increase with the addition of polymer. A correlation between average drop size and fluid viscosity was not observed. Both a relative relaxation time deduced from a simple die swell experiment and the first normal stress difference N₁correlated dissemination trials conducted at ambient temperatures. However, only the relative relaxation time correlation was consistent with breakup trials using heated fluids. Use of N₁, for predicting particle size from dissemination tests employing heated viscoelastic fluids can produce considerable error.     

Universal privacy‐preserving platform for SecaaS services

With the rapid growth of the Security‐as‐a‐Service market, concerns about privacy in exposing customer security policies to Cloud Service Providers have become critical. To resolve these issues, several solutions have been proposed over the past few years, each for a different kind of security service. However, as the number of security services outsourced into a cloud continues to grow, the need for a unified solution has become significant. This article introduces and presents a universal privacy‐preserving platform for SecaaS services that is based on a hybrid cloud architecture for maintaining the confidentiality of the customer's security policy. It is shown that this platform can be applied to all security services whose security policies can be represented in the form of a decision tree. This includes the vast majority of existing cloud‐based security services. With the small number of computationally‐expensive operations performed in a private cloud, the solution also does not require the implementation of a performant security engine on the customer's premises, allowing full advantage to be taken of private cloud offloading. It is also shown that the platform achieves better performance results than other existing solutions of this type. These findings were confirmed by experimental results.     

Human-robot collaboration in precise positioning of a three-dimensional object

This paper deals with fundamental issues of human-robot cooperation in precise positioning of a flat object on a target. Based on the analysis of human-human interaction, two cooperation schemes are introduced. Several algorithms implementing these schemes are developed. A general theoretical framework for human/robot cooperation has been developed to represent these algorithms. The evaluation of the algorithms was carried out using our in-house made robot prototype and experiments by human subjects has demonstrated the effectiveness of our schemes. The main problem was the regulation of the robot-human interaction. Since the robot has no range sensors, it has to rely on the force and displacement information resulting from the interaction with human to understand human intention. The way the robot interprets these signal is crucial for smooth interaction. To be able to carry out a concrete task a simplification was made, in which robot and human do not directly hold the object but a frame to which the object and various sensors are attached.     

Backscattered light confocal imaging of intracellular MTT-formazan crystals

Metabolically active animal and plant cells reduce MTT tetrazolium salt to a corresponding nonfluorescent formazan. Reduction of MTT by viable cells is exploited in a number of tests widely used in biological research. The aim of this study was to optimize a microscopy method of detecting small crystals of MTT-formazan formed in intact cells maintained in in vitro cultures. We examined scattering properties of small intracellular crystals of MTT formazan and found that the efficiency of light scattering was dependent on wavelength. Small (<3 microm) crystals of MTT-formazan, formed in viable cells, scattered red, but not blue, light. Large crystals, which are formed later at a stage when cells begin to lose viability, scattered both red and blue light. We conclude that optimal detection of early stages of crystallization of MTT-formazan in living cells is possible using confocal microscopy of red, but not blue, scattered light. High contrast and resolution of images can be achieved by filtering out interference effects in the frequency domain.     

Increasing microscopy resolution with photobleaching and intensity cumulant analysis

Super‐resolution fluorescence microscopy and its applications for analysis of biological structures are evolving rapidly field. A number of approaches aimed at overcoming the fundamental limit imposed by diffraction have been proposed in recent years. Here we present a modification of super‐resolution optical fluctuation imaging (SOFI), a technique based on spatio‐temporal evaluation of the optical signal from independently fluctuating emitters. Instead of rapid, reversible photoswitching, photobleaching is used to produce irreversible transitions between emitting and nonemitting states of the fluorochrome molecules. Simulated images are used to demonstrate that, in the absence of noise, the proposed SOFI modification increases the efficiency of transfer of high spatial frequencies in a fluorescence microscope. Correspondingly, a decrease of the point spread function (PSF) width is obtained. Moreover, the modified SOFI algorithm is capable of resolving point emitters in the presence of simulated noise. Using real biological images we demonstrate that an increase of resolution is obtained in 2D optical sections through densely packed chromatin in cell nuclei and lamin layer at the nuclear envelope. Finally, the approach is extended to 3D wide‐field microscopy, allowing reduction of out‐of‐focus image blurring. Microsc. Res. Tech. 78:958–968, 2015. © 2015 Wiley Periodicals, Inc.     

Viscoelastic breakup in a high velocity airstream

Viscoelastic fluids were injected into a high velocity airstream (200 m/s) to investigate how the addition of small polymer quantities to fluids significantly increase the resultant disseminated drop size. For each liquid tested several hundred resultant drops were sampled and measured using an automated image analyzer. The resultant mass median diameter (MMD) for a viscoelastic fluid was an order of magnitude larger than a comparable viscous Newtonian fluid. A relaxation time measured from a die swell experiment correlates the dissemination results suggesting, an elongational rather than shear breakup mechanism.