Polarization confocal microscopy and Congo Red fluorescence: a simple and rapid method to determine the mean cellulose fibril orientation in plants

The mean or net preferential orientation of cellulose fibrils in plant cell walls is detected with polarization confocal laser scanning microscopy using the fluorescence dichroism of Congo Red. Single cells, arrays of cells in a tissue, or the epidermis of whole organs can be assayed in vivo . Aerial parts require an extra pectinase treatment because of the cuticle, which is impermeable to aqueous solutions. Peeling off the epidermis can be an elegant alternative, especially for leaves. With this method the net preferential fibril orientation can be related to the symmetry axis of the cell in quantitative terms. Data issuing from this approach are useful in current research on plant biomechanics.     

Single-molecule force spectroscopy and imaging of the vancomycin/D-Ala-D-Ala interaction

The clinically important vancomycin antibiotic inhibits the growth of pathogens such as Staphylococcus aureus by blocking cell wall synthesis through specific recognition of nascent peptidoglycan terminating in D-Ala-D-Ala. Here, we demonstrate the ability of single-molecule atomic force microscopy with antibiotic-modified tips to measure the specific binding forces of vancomycin and to map individual ligands on living bacteria. The single-molecule approach presented here provides new opportunities for understanding the binding mechanisms of antibiotics and for exploring the architecture of bacterial cell walls.     

Towards a complementary balanced energy harvesting solution for low power embedded systems

The specific technical challenges associated with the design of an ambient energy powered electronic system currently requires thorough knowledge of the environment of deployment, energy harvester characteristics and power path management. In this work, a novel flexible model for ambient energy harvesters is presented that allows decoupling of the harvester’s physical principles and electrical behavior using a three dimensional function. The model can be adapted to all existing harvesters, resulting in a design methodology for generic ambient energy powered systems using the presented model. We also present a solution for the mathematical problem involved with the optimization of generator sizes when more than two harvesters are used, and demonstrate the ease of use of this solution for implementations on embedded systems with few system resources. Concrete examples are included to demonstrate the versatility of the presented design in the development of electronic appliances on system level.     

The cascading neural network: building the Internet of Smart Things

Most of the research on deep neural networks so far has been focused on obtaining higher accuracy levels by building increasingly large and deep architectures. Training and evaluating these models is only feasible when large amounts of resources such as processing power and memory are available. Typical applications that could benefit from these models are, however, executed on resource-constrained devices. Mobile devices such as smartphones already use deep learning techniques, but they often have to perform all processing on a remote cloud. We propose a new architecture called a cascading network that is capable of distributing a deep neural network between a local device and the cloud while keeping the required communication network traffic to a minimum. The network begins processing on the constrained device, and only relies on the remote part when the local part does not provide an accurate enough result. The cascading network allows for an early-stopping mechanism during the recall phase of the network. We evaluated our approach in an Internet of Things context where a deep neural network adds intelligence to a large amount of heterogeneous connected devices. This technique enables a whole variety of autonomous systems where sensors, actuators and computing nodes can work together. We show that the cascading architecture allows for a substantial improvement in evaluation speed on constrained devices while the loss in accuracy is kept to a minimum.     

Towards a nanoscale view of fungal surfaces

In the past years, atomic force microscopy (AFM) has offered novel possibilities for exploring the nanoscale surface properties of fungal cells. For the first time, AFM imaging enables investigators to visualize fine surface structures, such as rodlets, directly on native hydrated cells. Moreover, real-time imaging can be used to follow cell surface dynamics during cell growth and to monitor the effect of molecules such as enzymes and drugs. In fact, AFM is much more than a microscope in that when used in the force spectroscopy mode, it allows measurement of physicochemical properties such as surface energy and surface charge, to probe the elasticity of cell wall components and macromolecules, and to analyse the force and localization of molecular recognition events.     

Exploring the molecular forces within and between CbsA S-layer proteins using single molecule force spectroscopy

We used single molecule atomic force microscopy (AFM) to gain insight into the molecular forces driving the folding and assembly of the S-layer protein CbsA. Force curves recorded between tips and supports modified with CbsA proteins showed sawtooth patterns with multiple force peaks of 58+/-26pN that we attribute to the unfolding of alpha-helices, in agreement with earlier secondary structure predictions. The average unfolding force increased with the pulling speed but was independent on the interaction time. Force curves obtained for CbsA peptides truncated in their C-terminal region showed similar periodic features, except that fewer force peaks were seen. Furthermore, the average unfolding force was 83+/-45pN, suggesting the domains were more stable. By contrast, cationic peptides truncated in their N-terminal region showed single force peaks of 366+/-149pN, presumably reflecting intermolecular electrostatic bridges rather than unfolding events. Interestingly, these large intermolecular forces increased not only with pulling speed but also with interaction time. We expect that the intra- and intermolecular forces measured here may play a significant role in controlling the stability and assembly of the CbsA protein.     

Chemical force microscopy of single live cells

Traditionally, cell surface properties have been difficult to study at the subcellular level, especially on hydrated, live cells. Here, we demonstrate the ability of chemical force microscopy to map the hydrophobicity of single live cells with nanoscale resolution. After validating the technique on reference surfaces with known chemistry, we probe the local hydrophobic character of two medically important microorganisms, Aspergillus fumigatus and Mycobacterium bovis, in relation with function. Applicable to a wide variety of cells, the chemically sensitive imaging method presented here provides new opportunities for studying the nanoscale surface properties of live cells and for understanding their roles in mediating cellular events.     

Characteristics of High Cell Density Fermentations with Different Lager Yeast Strains

To improve the productivity of the beer fermentation process, several strategies can be adopted. One of these promising strategies could be the increase of suspended yeast cells in the reactor. Therefore, the fermentation characteristics of 11 lager yeast strains were studied in normal pitched worts (20 × 10⁶ cells/ mL) (LD) and in worts with a four‐fold higher pitching rate (HD). The fermentation rate was 2–4 times increased when high initial cell levels were used. The net yeast growth was somewhat similar between the LD and the HD fermentations, although the FAN uptake level was about 35% higher in the HD fermentations compared with LD. High viabilities were observed throughout the fermentations with high cell loadings. HD fermentations resulted in higher concentrations of all the measured fusel alcohols and higher maxima and residual concentrations of total diacetyl were observed. In contrast, higher levels of most of the esters were found at the normal pitching rate, although the results of isoamyl acetate were not significant. With the help of “Principal Component Analysis”, it became clear that the cell density had an important influence on the flavour profile, but that yeast specific preferences could not be overlooked as they determined the sensitivity of the yeast to the application of higher cell densities.     

Nanomicrobiology

Recent advances in atomic force microscopy (AFM) are revolutionizing our views of microbial surfaces. While AFM imaging is very useful for visualizing the surface of hydrated cells and membranes on the nanoscale, force spectroscopy enables researchers to locally probe biomolecular forces and physical properties. These unique capabilities allow us to address a number of questions that were inaccessible before, such as how does the surface architecture of microbes change as they grow or interact with drugs, and what are the molecular forces driving their interaction with antibiotics and host cells? Here, we provide a flavor of recent achievements brought by AFM imaging and single molecule force spectroscopy in microbiology.