Vilém Neděla

In Situ Study of Live Specimens in an Environmental Scanning Electron Microscope

In this paper we introduce new methodology for the observation of living biological samples in an environmental scanning electron microscope (ESEM). The methodology is based on an unconventional initiation procedure for ESEM chamber pumping, free from purge-flood cycles, and on the ability to control thermodynamic processes close to the sample. The gradual and gentle change of the working environment from air to water vapor enables the study of not only living samples in dynamic in situ experiments and their manifestation of life (sample walking) but also its experimentally stimulated physiological reactions. Moreover, Monte Carlo simulations of primary electron beam energy losses in a water layer on the sample surface were studied; consequently, the influence of the water thickness on radiation, temperature, or chemical damage of the sample was considered.

The low-temperature method for study of coniferous tissues in the environmental scanning electron microscope

The use of non‐standard low‐temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low‐temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low‐temperature method. An influence of the low‐temperature method on sample stability was also studied. The results indicate that high‐moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long‐time observation at different operation parameters. We finally show high applicability of the low‐temperature method on different types of conifers and Oxalis acetosella. Microsc. Res. Tech., 78:13–21, 2015.

Observation of a Brine Layer on an Ice Surface with an Environmental Scanning Electron Microscope at Higher Pressures and Temperatures

Observation of a uranyl-salt brine layer on an ice surface using backscattered electron detection and ice surface morphology using secondary-electron detection under equilibrium conditions was facilitated using an environmental scanning electron microscope (ESEM) at temperatures above 250 K and pressures of hundreds of Pa. The micrographs of a brine layer over ice grains prepared by either slow or shock freezing provided a complementary picture of the contaminated ice grain boundaries. Fluorescence spectroscopy of the uranyl ions in the brine layer confirmed that the species exists predominately in the solvated state under experimental conditions of ESEM.

Controlled dehydration of a biological sample using an alternative form of environmental SEM.

In this study a non‐conductive biological sample is observed free of charging artefacts when placed on a cooled Peltier stage in the specimen chamber of an alternative form of the environmental scanning electron microscope, equipped with a specially designed hydration system. This system was used to create dynamically changing surrounding conditions leading to controlled dehydration of the sample enabling us to visualize the topographical structure of a rat tongue in the transition region between the liquid and the gas state of water in the microscope specimen chamber.

Imaging of early conifer embryogenic tissues with the environmental scanning electron microscope

This article describes the usage of non-commercial environmental scanning electron microscope (ESEM) for the visualization of plant extracellular matrix in Abies alba and Abies numidica. Non sputter-coated samples free of using the common fixation technique observed at the relatively low humidity of the air environment with the pressure 550 Pa and the low temperature of the sample from −18 to −22°C give surprisingly very good results that show the natural structure of the tissues. This seems to be generally applicable. Moreover, a specially designed ionization detector of secondary electrons and a YAG:Ce3+ detector of backscattered electrons were used for better comparison.

Scintillation SE detector for variable pressure scanning electron microscopes

We present results obtained with a new scintillation detector of secondary electrons for the variable pressure scanning electron microscope. A detector design is based on the positioning of a single crystal scintillator within a scintillator chamber separated from the specimen chamber by two apertures. This solution enables us to decrease the pressure to several Pa in the scintillator chamber while the pressure in the specimen chamber reaches values of about 1000 Pa (7.5 Torr). Due to decreased pressure, we can apply a potential of the order of several kV to the scintillator, which is necessary for the detection of secondary electrons. Simultaneously, the two apertures at appropriate potentials of the order of several hundreds of volts create an electrostatic lens that allows electrons to pass from the specimen chamber to the scintillator chamber. Results indicate a promising utilization of this detector for a wide range of specimen observations.

Biooxidation of 2-phenylethanol to phenylacetic acid by whole-cell Gluconobacter oxydans biocatalyst immobilized in polyelectrolyte complex capsules

Abstract A high-performance biocatalyst in the form of encapsulated cells of Gluconobacter oxydans have been developed for production of phenylacetic acid (PAA) as a natural flavor component. Polyelectrolyte complex (PEC) capsules consisting of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl2, and NaCl were used for highly controlled and mild encapsulation of cells. Utilization of encapsulated G. oxydans cells was a significant improvement on existing data on operational stability of cells and cumulative product concentration during biocatalytic production of PAA from 2-phenylethanol. Concerning operational stability, encapsulated cells were active over 12 cycles with a high biotransformation rate, while free cells were inactive after 7 cycles of use. The biocatalytic properties of encapsulated G. oxydans were tested in a bubble column reactor over 7 days with a final cumulative product concentration of 25 g/L. High cell viability (90%) was observed within PEC capsules by confocal laser scanning microscopy, performed before and after repetitive PAA production in the bubble column reactor. The surface microstructure of fully hydrated capsules with and without G. oxydans cells was investigated and compared using an environmental scanning electron microscope.

Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications

Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.

High-efficiency detector of secondary and backscattered electrons for low-dose imaging in the ESEM

A new Combined System for high-efficiency detection of Secondary and Backscattered Electrons (CSSBE) in the ESEM consists of three detectors: an ionisation SE detector, an improved scintillation BSE detector, and a new Ionisation Secondary Electron Detector with an electrostatic Separator (ISEDS). The ISEDS optimizes conditions for electron-gas ionisation phenomena in the ESEM to achieve a strongly amplified signal from the secondary electrons with a minimal contribution from backscattered and beam electrons. For this purpose, it is originally equipped with an electrostatic separator, which focuses signal electrons towards a detection electrode and controls the concentration of positive ions above the sample. The working principle of the ISEDS is explained by simulations of signal electron trajectories in gas using the EOD program with our Monte Carlo module. The ability to detect the signal electrons in a selected range of energies is described with Geant4 Monte Carlo simulations of electron-solid interactions and proven by experimental results. High-efficiency detection of the ISEDS is demonstrated by imaging a low atomic number sample under a reduced beam energy of 5 keV, very low beam currents of up to 0.2 pA, and gas pressure of hundreds of Pa.

Imaging of native early embryogenic tissueof Scots pine (Pinus sylvestris L.) by ESEM

Abstract Environmental scanning electron microscopy enables the investigation of uncoated pine early embryogenic tissue samples in situ. The samples were examined under low vacuum conditions (air pressure 550 Pa) at a temperature of around -18°C by the AQUASEM II noncommercial environmental scanning electron microscope. The native extracellular matrix surface network was imaged by the environmental scanning electron microscope and in dark field mode of the optical microscope too. The backscattered electron detector disclosed brightness loci in the cells of early embryogenic culture. This work shows images of native pine embryogenic tissues. The continuity of extracellular matrix with structural integrity of plant organism is discussed.