AV ČR

Laboratory of advanced electron microscopy

Address

AV ČR
Královopolská 147
61264 Brno

www.isibrno.cz
laem.isibrno.cz

Contacts

Head

Tomáš Radlička
Phone: +420541514294
E-mail: radlicka@isibrno.cz

Contact person for users

Vladislav Krzyžánek
Phone: +420541514302
E-mail: vlk@isibrno.cz

Microscopy laboratories of ISI CAS are equipped with technical facilities and scientific know-how for imaging, elemental, and spectral analysis of hydrated samples using cryogenic scanning electron microscopy (cryo-SEM). Cryo-SEM finds its applications in the field of biological samples analyses (e.g. determination of foreign particles in cells, the study of intracellular granules,...). The combination with Raman spectroscopy enables complex analysis of several features of the cells fixed in the state closest to the native one, thanks to the most advanced vitrification techniques including high-pressure freezing.
Except for cryo-SEM, microscopy laboratories also offer quantitative analyses and diffraction imaging in STEM. Diffraction techniques enable imaging of phase contrast with maintaining high resolution, which finds its use in the study of light biological samples. The observation of samples is realized by a defocused beam, which reduces radiation damage. The final image is then obtained by processing the data of a 2D pixelated detector using ptychographic reconstruction.

Expertise and support provided to users

The well-experienced team at the laboratory of advanced electron microscopy provides conventional chemical preparation of biological samples using chemical fixative and dehydration agents for room temperature microscopy. Also, the facility is equipped with devices enabling cryogenic fixation using advanced techniques of high-pressure freezing followed by freeze-substitution for subsequent TEM imaging or freeze fracturing and etching for cryo-SEM observation, therefore users could use the expertise of trained specialists in the field of cryogenic analysis. In addition, imaging of samples is provided with possibilities of elemental analysis using energy-dispersive X-ray analyses or cathodoluminescence. SEM Magellan is equipped with a cryo stage for observation in temperatures of liquid nitrogen and was recently extended for cooling with liquid helium for observation at temperatures of -30K, thanks to this we can also offer such extreme conditions for biological specimens investigation. Cryo-SEM is usually extended with Raman spectroscopy of studied microbial cells enabling not only morphological studies but also chemical analyses of eg. intracellular granules, pigments or other components. The team is well experienced in quantitative imaging using a combination of various detected signals in SEM including thickness and mass-loss measurements by STEM.

Imaging technologies available

  • Raman Spectroscopy
  • Electron Microscopy
  • Correlative Light Electron Microscopy (CLEM)
  • Other

Equipment available (technical specification)

Electron microscopy

SEM Magellan 400/L (ThermoFisher)

  • Cryo-stage with adapter VCT100 (Leica microsystems) with optional extension to cooling down to -30K using liquid Helium
  • EDX detector Octane (EDAX)
  • CL detector MonoCL4+ (Gatan)
  • CL detector SPARC (Delmic)

High-pressure-freezer EM ICE (Leica microsystems)

Freeze-substitution unit EM AFS2 (Leica microsystems)

Cryo-vacuum chamber ACE600 (Leica microsystems)

Cryo-vacuum chamber BAF060 (Leica microsystems)

Raman microscopy

Raman microspectrometer inVia (Renishaw)

Raman tweezers

Research topics directly investigated in the CF

The research topics of the core facility are concentrated on the main research groups involved in the laboratory:

  • Microscopy for Biomedicine (Quantitative measurements of degradation/radiation damage of sensitive materials like embedding resins, ultrastructural studies of microorganisms producing biopolymers, instrumental and methodological research of scanning electron microscopy at very low temperature below the temperature of liquid nitrogen)
  • Electron Optics (data analysis in 2D STEM, development in ptychography)
  • Biophotonics and optofluidics (methodological development in the field of Raman spectroscopy and optical tweezers focused on the research of microorganisms producing biopolymers, phage therapy)

Therefore, the research contains instrumental and methodological developments on cryo-SEM, sample preparation especially for cryo-electron microscopy, quantitative imaging in SEM/STEM, and Raman spectroscopy.

External access to the equipment

The facility supports two modalities of access:
“Full-service mode” is the primary service modality.
“Self-service mode” is available only for selected services after training. The user is then obliged, prior to commencement, to sign the Agreement on Usage of the CF in view to fully comply with the safety regulations and accept the responsibility for the possible damage caused by the user during the measurement, or in connection with it.
For further details, please contact the contact person.

Courses

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Training materials (tutorials, handbooks etc.)

Following protocols and guidelines are in preparation and will be published on our website in the near future:
• protocol for room temperature chemical sample preparation for either SEM or TEM
• protocol for cryogenic sample preparation (high pressure freezing followed by freeze substitution/freeze fracturing and etching)
• conventional room temperature SEM imaging guideline (extended with EDX and cathodoluminescence)
• room temperature STEM analysis guideline
• cryo-SEM imaging guideline
• metal and carbon coating at cryogenic temperature guideline using ACE600 and BAF060
• protocol for TEM/STEM grids preparation (holey/leacy carbon film)