Standard Epon embedding and sectioning of cells and tissue.
The samples are prepared for electron microscopy in a process where we fix, dehydrated and infiltrated the cells with epon. We add contrast in the form of heavy metals and use an ultra microtome to cut thin sections (between 50-150 nm), suitable for the microscopy technique we want to use.
Cryo embedding, sectioning and labeling.
The samples are prepared for electron microscopy in a process where we fix the cells, and infiltrated the cells with sucrose and gelatin before they are frozen down in liquid nitrogen. We use a cryo ultra microtome to cut thin sections, before we label the sections with antibodies and small gold particles as markers.
CLEM, correlative light and electron microscopy.
CLEM combines the two microscopy platforms: light (or fluorescence) microscopy (LM) and electron microscopy (EM). The advantage of LM is that it can provide wide field images of whole cells, but its resolution is limited. Importantly light microscopy does not provide us with any useful structural information. The advantage of EM is that it can provide much higher resolution images, but only over specific regions of a cell at a time and not in live cells. CLEM combines the advantages of both techniques, allowing us to spot cellular structures and processes of interest in whole cell images with LM and then zoomed in for a further evaluation for EM. The EM analysis can off course contain further steps such as immuno-EM and electron tomography.
Electron tomography is an electron microscopy technique for obtaining a 3-D structural information. Images from regular utltrathin EM sections (100-150 nm) are collected at different tilt angles with the transmission electron microscope and this information is reassembled to yield a 3-D information. Reconstruction of this information is dependent on the use of Fourier-transformation algorithms and back projection. We currently using a 120 kV microscope for that purpose, using FEI-explorer software in combination with eTOMO ad IMOD, programs for reconstruction and modeling, respectively.