THEMATIC RESEARCH FOCUS

Group of environmental electron microscopy (EEM) Department of electron microscopy

Institute of Scientific Instruments of the CAS v.v.i.Czech Academy of Sciences

Královopolská 147, 612 64 Brno, Czech Republichttp://eem.isibrno.czVilém Neděla, Ing., Ing., Ph.D. / +420 541 514 333 / vilem@isibrno.cz

Live Mite observed in the ESEM AQUASEM II

RESEARCH AREA

/ Environmental scanning electron microscopy (ESEM) / Scanning electron microscopy (SEM) / Environmental and scanning transmission electron micros-

copy (Wet-STEM/STEM) / Signal electron and photon detection systems / Energy dispersive X-Ray spectroscopy / Computer based simulations for ESEM/SEM / Micro & nano technologies / Advanced electron microscopy of nano-materials (micro-

manipulation, gas/liquid injection, EBIC) / Dynamical in-situ/in-vivo study of live science samples / Study of nonconductive, wet and liquid samples

Laboratory of environmental electron microscopy in with high resolution environmental scanning electron microscope QUANTA 650 FEG (left) equipped with micromanipulators (right)

MISSION

To be the world leader in the low dose/energy environmental scanning electron microscopy and at the forefront in the field of static or dynamic in-situ characterisation of low emissive, beam sensitive, mostly non-conductive wet samples observed in their native state. To invent new methods, instrumentation and applications for further exploration of the nano-world.

Outer and inner side of wet polyvinyl-alcohol lances (LentiCatTM) in native state in ESEM

Non coated zinc oxide nanoparticles in ESEM

5 µm ___________

200 µm ______________ 400 nm ______________

EXCELLENCE

/ Low dose micro-morphological characterisation of untreated wet biological samples and polymers in ESEM / Dynamical in-situ observation of samples on the phase boundaries/transitions of matters and under well balanced thermo-

dynamic conditions in ESEM & electron microscopy for ice chemistry / Monte-Carlo simulations of electron-gas, electron-water and electron-solid interactions in ESEM and SEM / High-resolution imaging and X-ray micro-analysis of non/semi conductive samples in pressure conditions of ESEM / Research, development and manufacturing of very high efficient detectors of signal electrons for electron microscopy / Gas flow and heat transfer simulations for R&D of custom instrumentation for ESEM and SEM

Monte Carlo simulation of electron trajectories in gas (ESEM conditions)

Monte Carlo simulations of electron-solid sample interactions (C-Si multilayer)

New version of patented ISEDS with magnetic field (new detector for ESEM)

New scintillation BSE detector for SEM and ESEM

UP-TO-DATE ACTIVITIES

Research focus

/ Theoretical and experimental activities related to pushing boundaries of ESEM capabilities (Monte-Carlo simulations of electron-gas and electron-water interactions; simulations of gas flow/heat transfer for optimising observation condi-tions and development of new designs of electron micro-scopes, new detectors for possibility to detect weak/spatial signals with good signal to noise ratio as well as artefact free X-Ray particle analysis of untreated nonconductive wet sam-ples in ESEM)

/ Dynamical in situ characterisation of samples for explora-tion of time lapse processes and changes relating to external physical and chemical impacts, etc.

/ Morphological and structural analysis of untreated, uncoat-ed, unfrozen, hydrated or live samples

Main capabilitiesBasic research / Theoretical simulations of electron-gas, electron-water

and electron-solid interactions / Theoretical simulations of gas flow (pressure/velocity dis-

tribution) and heat transfer in ESEM / New method for the study of small live animals in ESEM / New method for long-term study and chemical analysis of

plant samples in ESEM (low temperature method for ESEM) / New method for repetitive observation of biological sam-

ples in ESEM

Applied research / Design and conversion of SEM Vega (Tescan) to experimental

ESEM AQUASEM II / Design and manufacturing of the new HAADF detector for

Hitachi TEM / Design and manufacturing of the new BSE detector for Jeol

SEM-JSM 5600LV (Gedeon Richter Co.) / Design and manufacturing of the new Scintillation detector

of SE for ESEM / R&D of the new „edge-free“ detector for Jeol SEM / R&D of the new YAP detector for Jeol SEM / Design and manufacturing of many prototypes of BSE detec-

tors (over 50 pieces) for Jeol, Hitachi & FEI

Innovations / New Ionisation SE detector with electrostatic separator for

ESEM (EU patent No. 2195822)

SUB-FIELDS OF GROUP ACTIVITIES

/ Micro/nanotechnology / Biology/biotechnology / Chemistry / Mineralogy / Archaeology / Semiconductor industry/Electronics / Pharmaceutical industry / Textile industry / Food industry / Building materials / Automotive / Photovoltaic and energy storage industry

Non coated surface of PUR with 10% CuO2 nanoparticles (ESEM for textile industry)

Fully expanded apple leaf, adaxial epidermis formed by puzzle-like cells (ESEM for plant biology)

Non coated SiO2 nanoparticles in epoxy resin (ESEM for micro/nano technology and Electronics)

Ice particle with pollutants (ESEM for ice chemistry)

Non coated buckle (ESEM for archaeology)

KEY RESEARCH EQUIPMENT

List of devices / Environmental scanning electron microscope AQUASEM II / High resolution environmental scanning electron microscope

QUANTA 650FEG / Very high sensitive scintillation BSE detectors(YAG,YAP and

CRY 18 scintillators) / Very high sensitive ionisation detectors for ESEM (ISEDS, Ioni-

zation SE detector) / Bruker X-Ray EDS analyser XFlash 6/60 + mapping (EDS micro-

analysis in SEM/ESEM) / Many types of special detectors (ET, GAD, LFD, GSED, ICD, DBS,

CL, etc) / Kleindiek micromanipulators + micro-gripper, low current

measurement kit + EBIC and RCI amplifier, gas and liquid mi-cro-injectors + special hydration system

/ Peltier cooling and heating stage (from -20°C to +1000°C) / Retractable solid state WetSTEM /STEM detector / Nav-CAM + three camber integrated IR cameras

3 µm ___________

100 µm ___________

1 µm ___________

100 µm ___________

7 mm ___________

ACHIEVEMENTS

AwardsKenbikyo Award 2016 – excellent paper of the journal for last two years (Japanese society of microscopy award). The best Ph.D. thesis (Czechoslovak Microscopy Society award), 2nd place in the event the Best doctoral thesis in the field of Building rehabil-itation and reconstruction (Scientific and Technical Association for Building Rehabilitation and Monument Preservation award).

Papers We are pushing the limits of possibilities of imaging and analysis of untreated, electrically non-conductive/semi-con-ductive and wet or liquid samples in ESEM. We are special-ised in the characterization of difficult to see and advanced materials using our developed new methods and unique instrumentation. We have published about 30 papers in im-pactful journals and about 130 contributions in conference proceedings or local journals.

/ Pioneering theoretical results of Monte Carlo simulations of electron-gas interactions and signal electron transporta-tion in gas owing to the collection efficiency of segmented ionization detector for signal electrons with selected ener-gies from units of eV to 18 keV.

V. Neděla, I. Konvalina, M. Oral, J. Hudec: The Simulation of En-ergy Distribution of Electrons Detected by Segmental Ioniza-tion Detector in High Pressure Conditions of ESEM. Microscopy and Microanalysis 21 (Suppl. 4), 264-269, 2015.

V. Neděla, I. Konvalina, M. Oral, J. Hudec: Monte Carlo Simulations of Signal Electrons Collection Efficiency and Devel-opment of New Detectors for ESEM. Microscopy and Microa-nalysis, 21 (Suppl 3), 1109-1110, 2015.

/ Significant theoretical work focused on calculations of higher-order deflections aberrations of the electron-optical system used for environmental scanning electron microsco-py and possibility of their dynamical corrections. This work indicates future trends in the ESEM.

M. Oral, V. Neděla, G. D Danilatos: Dynamic Correction of Higher-Order Deflection Aberrations in the Environ-mental SEM. Microscopy and Microanalysis 21 (Suppl. 4), 194-199, 2015.

/ World unique experimental results were attained by studying ice contamination processes at grain boundaries in environmentally compatible conditions of a high gas pres-sure and a relatively high temperature in a specially modi-fied ESEM in a combination with fluorescence microscopy.

J. Krausko, J. Runštuk, V. Neděla, P. Klán, D. Heger: Observation of a Brine Layer on an Ice Surface with an Environmental Scan-ning Electron Microscope at Higher Pressures and Tempera-tures. Langmuir 30 (No. 19), 5441-5447, 2014.

/ New method for high-resolution morphological study of plant samples in their native state. Method also allows to increase sample resistance to radiation damage by electron beam. This method was invented and experimentally tested on many types of plant biological samples in our laborato-ries.

V. Neděla, E. Tihlaříková, J. Hřib: The Low-Temperature Meth-od for Study of Coniferous Tissues in the Environmental Scan-ning Electron Microscope. Microscopy Research Technique 78, (No. 1), 13-21, 2015.

J. Hřib, B. Vooková, V. Neděla: Imaging of native early embryo-genic tissue of Scots pine (Pinus sylvestris L.) by ESEM. Open Life Sciences 10, (No. 1), 285-290. 2015.

V. Neděla, J. Hřib, L. Havel, J. Runštuk: Early state of spruce somatic embryos in native state observed using the ESEM and Cryo-SEM. Microscopy and Microanalysis 19, (s2), 20-21, 2013.

V. Neděla, J. Hřib, B. Vooková: Imaging of early conifer embryo-genic tissues with the environmental scanning electron micro-scope. Biologia Plantarum 56, (No. 3), 595-598, 2012.

/ Based on the results of Monte Carlo simulations of an electron beam with water involving study of free radicals concentration, a new, extremely sample-preserving method was introduced. It allows studying of small live and surviv-ing animals like mites in ESEM. The method presents basic research in the new field of live organism observation in the conditions of a high pressure, a low electron energy and a low beam current, utilizing advanced instrumentation sys-tems.

E. Tihlaříková, V. Neděla, M Shiojiri: In Situ Study of Live Speci-mens in an Environmental Scanning Electron Microscope. Mi-croscopy and Microanalysis 19, (No. 4), 914-918, 2013.

V. Neděla, E Tihlaříková, M. Shiojiri: New Environmental Scan-ning Electron Microscopy and Observation if Live Nature. Ken-bikyo. 49, (No. 1), 64-67, 2014.

/ Based on optimisation of the new method for study of live samples and in combination with our unique high effi-cient detectors of signal electrons for ESEM, a new method for morphological characterisation of very beam sensitive wet polyelectrolyte complex microcapsules containing sem-iliquid core with live cells was introduced. Our laboratory is the only one in the world able to observe these types of samples free of destruction or damage.

A. Bertóková, A. Vikartovská, M Bučko, P. Gemeiner, J. Tkáč, D. Chorvát, V. Štefuca, V. Neděla: Biooxidation of 2-phe-nylethanol to phenylacetic acid by whole-cell Gluconobac-ter oxydans biocatalyst immobilized in polyelectrolyte com-plex capsules. Biocatalysis and Biotransformation 33, (No. 2), 111-120, 2015.

A. Schenkmayerová, M. Bučko, P. Gemeiner, D. Treľová, J. Lacík, D. Chorvát Jr., P. Ačai, M. Polakovič, L. Lipták, M. Rebroš, M. Rosenberg, V. Štefuca, V.,Neděla, E. Tihlaříková: Physical and Bioengineering Properties of Polyvinyl Alcohol Lens-Shaped

PatentsUnique configuration of detector electrodes in combination with action of electric and magnetic fields allows to reach one of the world highest detection efficiency of the patented ISEDS. Among other unique benefits of the ISEDS is possibility to detect low energy signal electrons within optional energy range and under very low distances between the sample and final pressure limiting aperture of the ESEM so with very high signal to noise ration. Detector was invented and many years experimentally tested in our laboratories of environmental scanning electron microscopy.

New Ionisation SE detector with electrostatic separator for ESEM (CZ patent No. 299864, EU patent No. 2195822)For more details and publications see eem.isibrno.cz

Particles Versus Spherical Polyelectrolyte Complex Microcap-sules as Immobilisation Matrices for a Whole-Cell Baeyer–Vil-liger Monooxygenase. Applied Biochemistry and Biotechnolo-gy, 174 (No. 5), 1834-1849, 2014.

/ Advanced method for dynamical in-situ observation of untreated wet parasite in high gas pressure conditions and under reduced sample temperature was introduced. Unique results for taxonomical classification of parasites were pub-lished. This method opens up new possibilities for the use of environmental electron microscopy in the field of para-sitology.

Š. Mašová, E. Tihlaříková, V. Neděla: In Situ Dynam-ic ESEM Observations of Basic Groups of Parasites. In de Jonge, N. (ed.). Advances in Imaging and Electron Physics. Chapter One – CISCEM 2014: Proceedings of the Sec-ond Conference on In situ and Correlative Electron Microscopy. Vol. 190. Berlin : Elsevier, 2015, p. 92-95.

/ Scintillation SE detector for variable pressure and en-vironmental scanning electron microscopes. An original concept of a scintillator-photomultiplier detector of the secondary electrons which works in a wide range of pres-sures (10-4 Pa to 1000 Pa), the unique configuration capable of that in the world. Operation of the detector, its principle and its various configurations have been demonstrated in several experiments and published in impactful papers.

P. Čudek, J. Jirák, V. Neděla: Optimization of Signal Detection in Scintillation Secondary Electron Detector for ESEM and SEM. Microscopy and Microanalysis 19, (s2), 40-41, 2014.

Maxa, J.; Neděla, Vilém ; Jirák, Josef. ; Vyroubal, P. ; Hlad-ká, K. Analysis of gas flow in a secondary electron scintilla-tion detector for ESEM with a new system of pressure limit-ing apertures. Advances in Military Technology. 2012, roč. 7, č. 2, s. 39-44. ISSN 1802-2308.

Navicula radiosa imaged in native state by ESEM

EXPECTATIONS

OffersWe offer collaboration in the field of research and testing of a wide range of materials from electrically conductive to non-conductive dry, wet or liquid samples, special biologi-cal and polymeric samples (mostly free of any preparation). Investigation of morphological and structural changes of ma-terials under the influence of various external factors using unique non-commercial or improved commercial electron microscopes, advanced instrumentations and methods.

Partnership in local and international scientific or company projects.

Custom research, development and manufacturing of special detection systems and advanced instrumentation for electron microscopes.

Theoretical studies from the field of electron-gas/liquid/solid interactions and gas flow simulations for R&D of environmen-tal electron microscopes.

RequirementsWe look for cooperation with academic partners as well as companies in the fields of electron microscopy, physical, life and applied sciences as biology, chemistry, pharmacy, micro & nano-technologies, automotive, etc.

MAIN COLLABORATING PARTNERS

Collaboration with academic partners

University of Cambridge (Cambridge, GB) University of Kyoto (Kyoto, JP)

University of Nagoya (Nagoya, JP)Wroclaw University of Science and Technology (Wroclaw, PL)

University of technology Sydney (Sydney, AS)Charles University (Praha, CZ)

Brno University of Technology (Brno, CZ)Masaryk University (Brno, CZ)

Technical University of Liberec (Liberec, CZ)Mendel University (Brno, CZ)

Institute of Experimental Botany CAS (Praha, CZ)Institute of Botany CAS (Brno, CZ)

Institute of Chemistry, Slovak Academy of Sciences (Bratislava, SK)Institute of Macromolecular Chemistry, CAS (Praha, CZ)

IKEM (Praha, CZ)

Collaboration with companies

Hitachi High Technologies (Tokyo, JP)Jeol (Tokyo, JP)

FEI Czech Republic, s.r.o. (Brno, CZ)Tecpa s.r.o. (Brno, CZ)AutraDet (Brno, CZ)

BVT Technologies, a.s. (Brno, CZ)Contipro a.s. (Dolní Dobrouč, CZ)

Preciosa a.s. (Jablonec nad Nisou, CZ)Bosch spol. s.r.o. (České Budějovice, CZ)

Alkali-activated steel making slag in ESEM QUANTA FEG (Non coated surface)

5 µm ___________