Electron microscopy (EM) core facility

Biocenter Oulu Electron Microscopy Core Facility provides services and training in various electron microscopy techniques for the analysis of biological specimens. We also offer technical and scientific consultancy on experimental planning and provide training for sample preparation and operation of electron microscopes. With all the techniques EM analysis is optional. We are specialized in IEM and the ultrastructural analysis of gene-modified mouse tissues.

Our EM facility is part of the Biocenter Finland Electron Microscopy Technology Platform together with EM units from Helsinki and Kuopio.

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Information

Publications


Plastic embedding and thin sectioning

Specimen is fixed with either a mixture of 4 % paraformaldehyde and 1 % glutaraldehyde in 0,1 M phosphate buffer or 2 % glutaraldehyde in 0,1 M phosphate buffer, post-fixed with osmium tetroxide, dehydrated using increasing concentration of acetone or alcohol, and embedded in plastic resin (Epon). Ultrathin sections (70-80 nm) are post-stained with uranyl acetate and lead citrate before observation with TEM.

Thin sectioned epoxy embedded samples showing ultrastructure of E. coli, neuromuscular junction of mouse muscle, filtration barrier of mouse kidney glomerulus, and neuron of pig cerebral cortex.

High-pressure freezing and freeze substitution (HPF-FS)

One of the best available methods of biological sample preparation for electron microscopy as regards to structural preservation is cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method enables the observation of cells and cellular organelles in close-to-native state without the artefacts (extraction, coagulation, structural distortion) which are frequently observed when chemical fixation is used. A fresh cell or tissue sample is loaded together with filler or cryoprotectant on a sample holder and instantly frozen under very high pressure (2000 bar) using HPF machine. Freezing is followed by freeze substitution, which is done at temperature below -70°C, where water (in form of ice) in sample is replaced by organic solvent in presence of secondary fixative. After substitution samples can be embedded in plastic or rehydrated and processed by Tokuyasu technique for IEM.

Thin sectioned epoxy embedded samples of yeast S. cerevisiae (left) and mouse kidney (right) which were fixed using HPF-FS method.

Immunoelectron Microscopy (IEM)

For the localization of molecules at the ultrastructural level we apply Tokuyasu cryosectioning technique. Fresh specimen is fixed in 4 % paraformaldehyde in 0,1 M phosphate buffer, immersed in 2,3 M sucrose in PBS and frozen in liquid nitrogen. Thin cryosections are labeled with specific antibodies and conjugated gold markers are used to visualize antibody binding sites.

HPF-FS treated mouse kidney sample rehydrated and processed with Tokuyasu-method to show the localization of collagen IV in the basement membrane of kidney tubules (left). Cryosectioned mouse retina showing the localization of rhodopsin in rod cells (right).

Negative staining

Simple and rapid technique to analyze detailed structure of macromolecules, isolated organelles, and microorganisms by surrounding or embedding sample particles in an electron dense material that provides high contrast. A small droplet of sample is adsorbed on a carbon coated and glow-discharged grid. After excess sample is removed, grid is rinsed few times on a droplet of distilled water and placed on a droplet of stain (1% uranyl acetate or uranyl formate). After staining excess fluid is withdrawn and grid is dried before observation with TEM. It is also possible to combine immunostaining with negative staining. In that case antibody incubations are done prior the negative stain is applied.

Negatively stained actin filaments (left). Collagen fibers immunolabelled for decorin and stained with uranyl acetate (right).

Tomography

Electron microscopic tomography is a technique that enables the examination of three-dimensional biological structures. Thin specimen is tilted over a large angular range (typically ± 70°) around the tilt axis of goniometer stage and imaged at each tilt angle (usually at intervals of 1 or 2°). Obtained image set called tilt series is then processed to align images with each other and subsequently to reconstruct a three-dimensional volume. This volume is visualized as an image stack showing parallel slices through the volume. Direct volume visualization can be informative for some structures and analytical purposes. However, segmentation methods are often used with organelle structures for defining and dissecting components of the structure and to create a three-dimensional model which facilitates interpretation, the discovery of interrelationships, and measurement.

SEM Applications / Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM)

With this method it is possible to obtain series of images through conventionally prepared resin embedded tissue block. Aligned high resolution image series can then be used for ultrastructural volume reconstruction and 3D-analysis of cellular structures. In the dual beam electron microscope the focused gallium ion beam and electron source are arranged at an angle which allows the layer-by-layer removal of material from the specimen surface and subsequent imaging with scanning electron beam. Process is fully automated and once suitable area is located and milling and imaging parameters are set, instrument will automatically produce nearly aligned image series.

Orthogonal views of image stack obtained from mouse heart tissue using slice and view technique with FIB-SEM (left). 3D-model of a heart capillary, where endothelial cells (blue and cyan) forming the capillary and a pericyte (green) surrounding the capillary were segmented and surface rendered (right).

SEM Applications / Corrosion cast technique

Vascular corrosion casting is an excellent tool for morphological examination of organ and tissue microcirculation under normal and pathological conditions. In short, mice or rats are perfused and blood is replaced with a low viscosity resin. After resin has cured, KOH maceration is used to remove the surrounding tissue and to reveal the replicate of vasculature. Due to polyurethane-based resin (PU4ii) casts are highly elastic and the shape and volume of organs are preserved. Casts are normally imaged using SEM but can be further processed for analysis using confocal microscopy (resin is autofluorescent), micro-computed tomography (microCT), or optical projection tomography (OPT), which allows the production of 3D reconstructions for quantitative morphometric analysis.

Vascular corrosion cast of mouse kidney viewed under stereo microscope (left). SEM analysis of mouse kidney corrosion cast showing several tight capillary coils forming glomeruli (right).

Instruments:

  • Tecnai G2 Spirit 120 kV TEM with Veleta and Quemesa CCD cameras (BF-funding)
  • Sigma HD VP FE-SEM equipped with ET-SE and In-lens SE detectors, VPSE G3 detector for low vacuum mode, and 5Q-BSD detector

Location – The Main Building of Medical Campus, Aapistie 5A
(Tecnai room 467B, Sigma FE-SEM room 320A)

tel: +358-(0)294 486144, int. 48-6144 (Tecnai), 48-6146 (CM100)

Instrument reservation:

Instruments can be reserved using the Asimov reservation book:

Specimen preparation laboratory

Lab is equipped with modern instruments for the preparation of cells, organelles, and tissue samples for various EM analyses.

Equipment:

  • Leica EM UC7 ultramicrotome with EM FC7 cryo attachment and EM CRION ionizer
  • Leica EM UC6 ultramicrotome with EM FC6 cryo attachment
  • Leica ultracut UCT ultramicrotome
  • Leica ultracut E
  • EM-Pact high pressure freezing device (Leica)
  • Leica EM AFS2 freeze-substitution device with automatic liquid handling system (BF-funding)
  • Leica EM TP automated tissue processor
  • K850 Critical Point Dryer
  • Q150T ES Instrument with interchangeable inserts for sputter coating or carbon evaporation equipped with film thickness monitor and glow discharge insert

Location – EM laboratory, Biocenter Oulu laboratories in the Main Building of Medical Campus, Aapistie 5A
(room 488B, 4th floor)

tel: +358-(0)294 486144, int. 48-6144

Contact persons:

Ilkka Miinalainen, Coordinator
tel: +358-(0)294 486145, int. 48-6145
E-mail: ilkka.miinalainen (at) oulu.fi

Specimen preparation laboratory:
Sirpa Kellokumpu,  Tarja Piispanen, Päivi Tyni
tel: +358-(0)294 486144, int. 48-6144
E-mail: firstname.lastname (at) oulu.fi

Specimen fixation instructions:

Fixation of tissues for TEM and IEM

Fixation of cultured cells for TEM and IEM

Fixation of isolated organelles for TEM and IEM

Order Form:

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Prices:

Prices for academic groups

Prices for commercial users

Last updated: 21.3.2017