Techniques for Transmission Electron Microscopy (TEM)
Plastic embedding and thin sectioning
Conventional sample processing for transmission electron microscopy includes fixation with e.g. 2 % glutaraldehyde in 0,1 M phosphate buffer, post-fixation with osmium tetroxide, dehydration in ascending concentration series of alcohol, and embedding in plastic resin (Epon). Ultarmicrotome is used to cut ultrathin sections (70-80 nm) that are finally post-stained (contrasted) with uranyl acetate and lead citrate.
Ongoing studies, examples
- Assistant professor Arja Kullaa, Institute of Dentistry (Co-supervisor; DDS and BDS studies)
- Professor Kai Kaarniranta, School of Medicine/Ophthalmology (Collaboration)
- Postdoctoral researcher Mika Reinisalo, School of Pharmacy (Collaboration)
With the aim of avoiding artefacts caused by chemical fixation, the specimens can be fixed by using radip freezing. After freezing, the process is followed by freeze-substitution (FS) meaning that water within the specimen is substituted with organic solvent at low temperature (less than -70°C) and embedded in plastic or processed for IEM. Our aim is to develop this methodology for routine use in 2016.
This method is typically used to study e.g. isolated organelles, particles or viruses. In practice, a droplet of biological microorganism is incubated on a carbon-coated and glow-discharged TEM grid. Excess sample is removed and rinsed, contrasted using heavy element salts (e.g. uranyl acetate). Also immunostaining is applicable for the specimens.
- Academy research fellow Kirsi Rilla, Institute of Biomedicine
- PhD student Kai Härkönen: "Extracellular vesicles - Biogenensis, uptake and biological effects on cancer cells" (Co-supervisor)
- Postdoctoral researcher Jenni Karttunen, Dept. of Neurobiology (Collaboration)
- Associate professor Tarja Malm, Dept. of Neurobiology (Collaboration)
Equipped with energy dispersive X-ray spectroscopy (EDS), electron microscopy can be used to study elemental composition of the specimens at the same time with EM imaging. We have developed intrumentation and analysis methods especially for biological specimens.
Ongoing studies, examples
- PhD Minna Kivimäenpää, Dept. of Environmental Sciences(Collaboration); Cerium perhydroxide precipitates
- PhD Arja Tervahauta, Dept. of Biology (Collaboration)
In TEM, we have the possibility to carry out electron tomography (ET) studies. ET can be applied to study 3D structures even in nanometer scale. A series of images with changing tilt angle is captured during imaging and afterwards, 3D tomogram is reconstructed and analyzed.
- MSc study: Muhammad Arshad Haroon "3D Tomography in Transmission Electron Microscopy - Optimizing alignment using Affine Transform and Scale Invariant Feature Transform" (Supervisor)
- Professor Kai Kaarniranta, School of Medicine/Ophthalmology (Collaboration); Perinuclear drusen
- Assistant professor Arja Kullaa, Institute of Dentistry (Collaboration); Oral mucosa