Electron Microscopy

 

About

The Electron Microscopy Core Facility provides expertise and cutting edge equipment for a broad range of biological sample preparation and ultrastructural imaging techniques. The core facility deals with various biological samples: human and animal cell cultures, plant and animal tissues, worms, microorganisms, lipid micelles. The sample preparation techniques include routine chemical fixation and resin embedding, cryofixation using high-pressure freezing technique, freeze-substitution, plunge-freezing, cryosectioning, and immunolabeling, including simultaneous detection of multiple targets by our self-developed methods.

High-pressure freezing machine, two automatic freeze-substitution machines, cryo-ultramicrotomes, Vitrobot for automated plunge-freezing, as well as additional wet lab equipment are available. The core facility is equipped with two transmission electron microscopes (TEM) – a standard instrument for routine observation and an advanced 200 kV instrument providing the possibility of high-resolution TEM, 3D electron tomography, cryo-electron microscopy and electron energy-loss (EELS) analysis.

The team has a long expertise in the development and optimization of sample preparation techniques for electron microscopy. We have optimized the cryofixation of cultured cells and sample processing after cryofixation, developed a completely new system for simultaneous immunolabeling of five molecular targets, a special technique for combination of 3D pre-embedding immunolabeling and 2D on-section immunolabeling to provide more possibilities for spatial analysis of biological processes. Currently, we also collaborate with the companies developing instrumentation for electron microscopy to contribute in development of sample preparation for various modes of microscopic analysis.

We collaborate with visiting scientists from the Czech Republic, Croatia, France, Japan, and Switzerland. The following topics are covered: cellular mechanisms of infection by Francisella tularensis; heavy metals uptake by plant roots; mechanisms of apoptosis induction; regulation of p62 in dividing and terminally differentiated cells, mechanisms of spermatogenesis, cellular mechanisms of diabetes; analysis of antigen presence in viral vaccines, study of lipid micelles structure with respect to their suitability for DNA transfection, compartmentalization of actin-related proteins in the cell nucleus and their involvement in chromatin compartmentalization.

The Electron Microscopy Core Facility associated to the IMG Microscopy Centre is part of the IMG Czech-BioImaging node and Prague Euro-BioImaging node.

 

czech bio-imaging logo

 

Location IMG – building F, 1st floor, rooms 1.60, 1.52 and basement
01.155.1, 01.150, 01.152, 01.153, 01.154, 01.151
Phone +420 241 063 153
Documents How to properly acknowledge the IMG Microscopy Centre

 

People

Electron Microscopy Facility Team  

Head Vlada Philimonenko, M.Sc., Ph.D.
Technicians Zuzana Lubovská, M.Sc (deputy facility leader)

Dominik Pinkas, M.Sc.
Lab technician Ivana Nováková
PhD student Jana Schrenková, M.Sc. (maternity leave)

 

Instrumentation

 

Electron microscopes

FEI Tecnai G2 20 LaB6 transmission electron microscope

FEI Tecnai G2 20 LaB6 transmission electron microscope operated at 200 kV with tomography, cryostage and elemental mapping (EELS, GIF) equipped with Gatan Model 894 UltraScan 1000 camera (pixel size 14 x 14 µm)

The Tecnai G2 20 is a highly advanced state-of-the-art transmission electron microscope equipped with cryo-tomography stage, Gatan Model 914 High Tilt Tomography Cryo-Holder, Olympus Morada CCD camera and Gatan 863 Tridiem Imaging Filter (GIF). It offers high performance, versatility and ease of use. The accessories fitted onto this system are embedded into the user interface, allowing differently experienced operators to fully utilize the functionality of the total system. The machine is pre-set at 120 and 200kV, thus allowing to collect EM data from specimen for which either high contrast or high resolution is preferred. It gives a maximum resolution of 0.27 nm.

FEI Morgagni 268 Transmission Electron Microscope with Olympus Mega View III Digital Camera

FEI Morgagni 268 transmission electron microscope operated at 80 kV
equipped with Mega View III CCD camera (pixel size 6.45 x 6.45 µm)

Up to 100 kV TEM with a tungsten filament electron beam source and a side-mounted Olympus Mega View III CCD camera (pixel size 6.45 x 6.45 µm). The machine is running at 80kV, thus allowing to collect EM data from biological specimens for which high contrast is preferred to high resolution. The embedded Soft Imaging System (SIS) software performs the on-the-run image processing and enhancement allowing the immediate final-quality digital visualization of the sample. A maximum resolution of 0.45 nm is available along with magnification up to 200 000. The microscope is regularly used for routine sample screening and acquisition of the micrographs in publication quality.

 

Cryo-equipment for sample preparation

LEICA EMPACT2 high-pressure freezer preventing the formation of crystal ice

Sample Preparation for Electron Microscopy – LEICA EMPACT2: high-pressure freezing of samples preventing the formation of crystalic ice

The Leica EMPACT2 is a high pressure freezing system for vitrifying samples up to 200 µm in thickness without the artefacts of chemical fixation. It preserves the biological samples in near-to-native state for further high-resolution EM observation. Bayonet loading device provides automatic specimen orientation. After freezing, the specimen is ejected into liquid nitrogen bath. The temperature/pressure curve is displayed for each run allowing the precise control of the process. Internal memory allows to collect up to 8 000 freezing runs.

High-pressure frozen samples can be processed by either Frozen Hydrated Sectioning or Freeze Substitution. The Freeze Fracture Holder is also available.

LEICA EM AFS2 processing the samples in automated or manual mode for freeze-substitution and plastic embedding

Sample Preparation for Electron Microscopy – Leica EM AFS2: replacement of ice in the sample with resins

Leica EM AFS2 performs freeze substitution and progressive lowering of temperature, then allowing low temperature embedding and resin polymerization. It is equipped with UV lamp. Specimen can be visualized with the attached stereomicroscope under internal LED illumination. The temperature working range is from 140 °C to +70°C. The 35 l Dewar enables long protocols. Liquid nitrogen filling from outside of specimen chamber allows refilling during the run. The “Deep Freeze” function allows sample transfer at temperatures around -140 °C. Adjustable transfer function of gaseous (evaporated) liquid nitrogen excludes humidity and oxygen, which results in no contamination of the specimen.

Several substitution systems are available: Substitution-Capsule and Flat Embedding System, New Microtube Embedding System EM AFS2 and Sapphire Disc FS System.

The Leica EM FSP (freeze substitution processor) is an automatic reagent handling system. Mounted on the Leica EM AFS2, it dispenses reagents for both FS and PLT applications minimizing operators contact with toxic media and reducing risk to lose a specimen.

FEI Vitrobot Mark III

FEI Vitrobot Mark III
semi-automated cryo-plunger

A semi-automated cryo-plunger. It allows producing high-quality vitrified samples for single particle analysis or cryo-tomography research applications. It performs the cryo-fixation under the constant physical and mechanical conditions (temperature, relative humidity, blotting conditions and freezing velocity). This ensures reproducible high quality cryo-fixation results and sample preparation throughoutput for the following cryo-TEM observation.

 

Ultramicrotome

LEICA EM UltraCut6 cutting ultrathin (cryo-) sections 50 - 100 nm and semi-thick sections for tomography (200 – 500 nm)

Sample Preparation for Electron Microscopy – LEICA EM UltraCut6: cutting ultrathin (cryo)sections 50 – 100 nm

The Ultramicrotome Leica EM UC6 provides easy preparation of semi- and ultrathin sections as well as perfect, smooth surfaces of biological samples for TEM, SEM, AFM and LM examination. Eucentric movement of the stereomicroscope offers optimized positioning for approach with glass and diamond knives. It also helps in section observation at low water levels (e.g. for Lowycryls and dry sections) and cryosectioning without loss of ergonomic posture. Three independent brightness-controlled LED light sources provide illumination for toplight, backlight and specimen trans-illumination. The Integrated Antivibration System prevents influence from external vibrations. Motorized North-South and East-West movement of the knife stage in conjunction with the touch-sensitive control panel enables fast and safe alignment of knife and specimen with help files and prompts to hand for beginners (up to seven different user-settings can be stored). Programmable knife and cutting movements offer significant ease for trimming (Autotrim mode).

Accessories:

Low Temperature Sectioning System Leica EM FC6

Leica EM FC6 in combination with the Ultramicrotome Leica EM UC6 is designed for ultrathin cryosectioning of biological specimens in the range from -185 °C to -15 °C. Improved sectioning quality is provided with contact free through the wall specimen arm for vibration free sectioning, eucentric knife rotation with centre click stop from outside for easy alignment of knife and blockface, individual temperatures for specimen, knife and gas, automatic rapid cooling function to save time when cooling down to temperatures below -165°C. Specimen holder is locked with torque limited screw to provide optimum locking of sample without damage to the screw.

 

Accessory equipment

LKB KnifeMaker Type 7800 to prepare glass knives for ultramicrotomy

LKB KnifeMaker Type 7800

The classical LKB KnifeMaker produces perfect glass knives for semi-thin sections for EM and LM applications. The knife maker uses 6–7 mm thick and 25 or 38 mm wide glass strips.

E6700 Turbo Molecular Bench Top Vacuum Evaporator

E6700 Turbo Molecular Bench Top Vacuum Evaporator for TEM and SEM applications like producing carbon support films, preparation of replicas, shadowing, aperture cleaning, etc.

It enables transmission and scanning electron microscopy (TEM and SEM) specimen preparation, like producing carbon support films, preparation of replicas, shadowing, aperture cleaning, etc. The usage of a turbo molecular vacuum pump ensures oil free vacuum chamber environments. The further benefits are: simple operations, rapid pump down and high vacuum operation (routinely 10-5/10-6mbar). All accessory power supplies are vacuum interlocked for user protection and cannot be energized unless the chamber is under vacuum. Mounted on a 300 mm (12”) diameter stainless steel base plate, the TEM set includes resistive evaporation sources for 6 mm diameter carbon rods, metal evaporation (tungsten basket), tiltable specimen holder and base, glow discharge work chamber and baseplate electrode. The jigging is a one piece assembly making it easy to remove or modify. A “cool” sputtering head (not routinely mounted) allows SEM sputter coating. The base has 10 ports through which electrical, vacuum and water connections can be made. A rotating source shutter is also available. A 250 mm (10”) diameter bell jar provides the vacuum chamber.

 

Gold – software tools for ultrastructural studies

Electron microscopy (EM) is the method of choice to study subcellular compartments in biological samples with high resolution. In many cases, the cellular compartments are defined by the content of specific molecules or by a presence of specific functions and they are morphologically inconspicuous in EM preparations. Such compartments can be detected using antibodies with attached label, usually gold particles. In this case, however, it is necessary to delineate the borders of the immunogold clusters in order to define the topology of the compartment. However, the labeling has a point pattern, while a compartment is a coherent area. In addition, some background labeling is always present which complicates identification of the labeled compartments. Stereological image analysis is therefore required to interpret the density distribution of gold particles.

 

More information – http://nucleus.img.cas.cz/gold/.

 

 

Gallery

 

 

Financial Support

The activities of the Microscopy Centre – Core Facility for Electron Microscopy are supported from the program for large research infrastructures of the Ministry of Education, Youth and Sports within the project “National Infrastructure for Biological and Medical Imaging (Czech-BioImaging – LM2015062)“.

 

Last change: January 16, 2019