Armin Zankel

Ultramicrotomy in the ESEM, a versatile method for materials and life sciences

We here present the results of the first materials science analyses obtained with the prototype of a serial block‐face sectioning and imaging tool, 3View™ of Gatan, Inc (Pleasanton, CA, U.S.A.). It is a specially designed ultramicrotome operating in situ within an environmental scanning electron microscope originally developed for life science research. The microtome removes thin slices from the sample and the environmental scanning electron microscope images each new block surface of the specimen (serial block‐face scanning electron microscopy). The Schottky emitter (FEG) of the microscope delivers high spatial resolution and has the advantage of stable performance and high durability. The slice thickness can typically be selected between 50 and 100 nm. It is possible to cut hundreds of slices and simultaneously acquire images with Digital Micrograph™ Model 700 (Gatan, Inc.). This article outlines the set‐up and describes the automated process. The preparation of specimens for in situ ultramicrotomy is explained and the parameters for good image quality are discussed. In addition, special operative and analytic features of the controlling software are presented. Three different technical materials and one botanical specimen were analyzed delivering first results of this method for materials science and for botany.

Reconstruction and Characterization of a Polymer-Based Monolithic Stationary phase using Serial Block-Face Scanning Electron Microscopy

Porous, polymer-based materials are increasingly used as stationary phases in separation science and catalysis, yet their morphology remains largely unknown. The main difficulty lies in reconciling their soft matter nature with the demands of microscopic imaging techniques. We analyze the morphology of a hyper-cross-linked poly(styrene-divinylbenzene) monolith in capillary column format from a sample volume of 60.5 × 60.5 × 19.9 μm(3) reconstructed by serial block-face scanning electron microscopy. To obtain a suitable specimen, the polymer skeleton was stained with tetraphenyllead and the void space filled with resin before the whole monolith was resin-embedded after removing the fused-silica capillary. Chord length distribution analysis revealed characteristic lengths of 7.32 and 0.73 μm, corresponding to two distinct macropore types. The macroporosity (77% on average) was found to increase systematically from the wall to the center. Our results provide valuable insights into the formation process of the monolith and its stationary-phase properties.

Serial sectioning methods for 3D investigations in materials science

A variety of methods for the investigation and 3D representation of the inner structure of materials has been developed. In this paper, techniques based on slice and view using scanning microscopy for imaging are presented and compared. Three different methods of serial sectioning combined with either scanning electron or scanning ion microscopy or atomic force microscopy (AFM) were placed under scrutiny: serial block-face scanning electron microscopy, which facilitates an ultramicrotome built into the chamber of a variable pressure scanning electron microscope; three-dimensional (3D) AFM, which combines an (cryo-) ultramicrotome with an atomic force microscope, and 3D FIB, which delivers results by slicing with a focused ion beam. These three methods complement one another in many respects, e.g., in the type of materials that can be investigated, the resolution that can be obtained and the information that can be extracted from 3D reconstructions. A detailed review is given about preparation, the slice and view process itself, and the limitations of the methods and possible artifacts. Applications for each technique are also provided.

Investigation of mircroorganisms colonising activated zeolites during anaerobic biogas production from grass silage.

The colonisation of activated zeolites (i.e. clinoptilolites) as carriers for microorganisms involved in the biogas process was investigated. Zeolite particle sizes of 1.0-2.5mm were introduced to anaerobic laboratory batch-cultures and to continuously operated bioreactors during biogas production from grass silage. Incubation over 5-84 days led to the colonisation of zeolite surfaces in small batch-cultures (500 ml) and even in larger scaled and flow-through disturbed bioreactors (28 l). Morphological insights were obtained by using scanning electron microscopy (SEM). Single strand conformation polymorphism (SSCP) analysis based on amplification of bacterial and archaeal 16S rRNA fragments demonstrated structurally distinct populations preferring zeolite as operational environment. via sequence analysis conspicuous bands from SSCP patterns were identified. Populations immobilised on zeolite (e.g. Ruminofilibacter xylanolyticum) showed pronounced hydrolytic enzyme activity (xylanase) shortly after re-incubation in sterilised sludge on model substrate. In addition, the presence of methanogenic archaea on zeolite particles was demonstrated.

A method to measure the total scattering cross section and effective beam gas path length in a low-vacuum SEM.

A method is presented to determine the total scattering cross section of imaging gases used in low-vacuum scanning electron microscopy or environmental scanning electron microscopy. Experimental results are presented for water vapor, nitrogen gas and ambient air for primary beam electron energies between 5 and 30 keV. The measured results are compared and discussed with calculated values. This method allows the effective beam gas path length (BGPL) to be determined. The variations of the effective BGPL with varying chamber pressure are presented.

The 3D pore structure and fluid dynamics simulation of macroporous monoliths: High permeability due to alternating channel width.

Polymethacrylate-based monoliths have excellent flow properties. Flow in the wide channel interconnected with narrow channels is theoretically assumed to account for favorable permeability. Monoliths were cut into 898 slices in 50nm distances and visualized by serial block face scanning electron microscopy (SBEM). A 3D structure was reconstructed and used for the calculation of flow profiles within the monolith and for calculation of pressure drop and permeability by computational fluid dynamics (CFD). The calculated and measured permeabilities showed good agreement. Small channels clearly flowed into wide and wide into small channels in a repetitive manner which supported the hypothesis describing the favorable flow properties of these materials. This alternating property is also reflected in the streamline velocity which fluctuated. These findings were corroborated by artificial monoliths which were composed of regular (interconnected) cells where narrow cells followed wide cells. In the real monolith and the artificial monoliths with interconnected flow channels similar velocity fluctuations could be observed. A two phase flow simulation showed a lateral velocity component, which may contribute to the transport of molecules to the monolith wall. Our study showed that the interconnection of small and wide pores is responsible for the excellent pressure flow properties. This study is also a guide for further design of continuous porous materials to achieve good flow properties.

Assessing Structural Correlations and Heterogeneity Length Scales in Functional Porous Polymers from Physical Reconstructions

A general, model-free, quantitative approach to the key morphological properties of a porous polymer monolith is presented. After 3D reconstruction, image-based analysis delivers detailed spatial and spatially correlated information on the structural heterogeneities in the void space and the polymer skeleton. Identified heterogeneities, which limit the monoliths performance in targeted applications, are traced back to the preparation process.

Activated zeolite--suitable carriers for microorganisms in anaerobic digestion processes?

Plant cell wall structures represent a barrier in the biodegradation process to produce biogas for combustion and energy production. Consequently, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required. Here, we show that natural activated zeolites (i.e. trace metal activated zeolites) represent eminently suitable mineral microhabitats and potential carriers for immobilisation of microorganisms responsible for anaerobic hydrolysis of biopolymers stabilising related bacterial and methanogenic communities. A strategy for comprehensive analysis of immobilised anaerobic populations was developed that includes the visualisation of biofilm formation via scanning electron microscopy and confocal laser scanning microscopy, community and fingerprint analysis as well as enzyme activity and identification analyses. Using SDS polyacrylamide gel electrophoresis, hydrolytical active protein bands were traced by congo red staining. Liquid chromatography/mass spectroscopy revealed cellulolytical endo- and exoglucanase (exocellobiohydrolase) as well as hemicellulolytical xylanase/mannase after proteolytic digestion. Relations to hydrolytic/fermentative zeolite colonisers were obtained by using single-strand conformation polymorphism analysis (SSCP) based on amplification of bacterial and archaeal 16S rRNA fragments. Thereby, dominant colonisers were affiliated to the genera Clostridium, Pseudomonas and Methanoculleus. The specific immobilisation on natural zeolites with functional microbes already colonising naturally during the fermentation offers a strategy to systematically supply the biogas formation process responsive to population dynamics and process requirements.

Bioresponsive systems based on polygalacturonate containing hydrogels

Polysaccharide acid (PSA) based devices (consisting of alginic acid and polygalacturonic acid) were investigated for the detection of contaminating microorganisms. PSA-CaCl(2) hydrogel systems were compared to systems involving covalent cross-linking of PSA with glycidylmethacrylate (PSA-GMA) which was confirmed with Fourier Transformed Infrared (FTIR) analysis. Incubation of PSA-CaCl(2) and PSA-GMA beads loaded with Alizarin as a model ingredient with trigger enzymes (polygalacturonases or pectate lyases) or bacteria lead to a smoothening of the surface and exposure of Alizarin according to Environmental Scanning Electron Microscopy (ESEM) analysis. Enzyme triggered release of Alizarin was demonstrated for a commercial enzyme preparation from Aspergillus niger and with purified polygalacturonase and pectate lyase from S. rolfsii and B. pumilus, respectively. In contrast to the PSA-CaCl(2) beads, cross-linking (PSA-GMA beads) restricted the release of Alizarin in absence of enzymes. There was a linear relation between release of Alizarin (5-348 μM) and enzyme activity in a range of 0-300 U ml(-1) dosed. In addition to enzymes, both PSA-CaCl(2) and PSA-GMA beads were incubated with Bacillus subtilis and Yersinia entercolitica as model contaminating microorganism. After 72 h, a release between 10 μM and 57 μM Alizarin was detected. For protection of the hydrogels, an enzymatically modified PET membrane was covalently attached onto the surface. This lead to a slower release and improve long term storage stability based on less than 1% release of dye after 21 days. Additionally, this allowed simple detection by visual inspection of the device due to a colour change of the white membrane to orange upon enzyme triggered release of the dye.

Synaptic Connections of First-Stage Visual Neurons in the Locust Schistocerca gregaria Extend Evolution of Tetrad Synapses Back 200 Million Years

The small size of some insects, and the crystalline regularity of their eyes, have made them ideal for large‐scale reconstructions of visual circuits. In phylogenetically recent muscomorph flies, like Drosophila, precisely coordinated output to different motion‐processing pathways is delivered by photoreceptors (R cells), targeting four different postsynaptic cells at each synapse (tetrad). Tetrads were linked to the evolution of aerial agility. To reconstruct circuits for vision in the larger brain of a locust, a phylogenetically old, flying insect, we adapted serial block‐face scanning electron microscopy (SBEM). Locust lamina monopolar cells, L1 and L2, were the main targets of the R cell pathway, L1 and L2 each fed a different circuit, only L1 providing feedback onto R cells. Unexpectedly, 40% of all locust R cell synapses onto both L1 and L2 were tetrads, revealing the emergence of tetrads in an arthropod group present 200 million years before muscomorph flies appeared, coinciding with the early evolution of flight. J. Comp. Neurol. 523:298–312, 2015.