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Types, techniques and services in electron microscopy
Our microscopy unit has two electron microscopes:
- transmission TEM JEOL 1400plus and
- scanning SEM Zeiss Sigma VP. The SEM can be used in the 3-View mode, serial block-face SBF-SEM.
TEM Transmission Electron Microscopy
Exploring subcellular structure in ultra-thin specimens
TEM is usually performed on fixed, osmicated (treated with osmium tetroxide), completely dehydrated specimens. They are infiltrated with epoxy resins, cooked into hard blocks and cut into 50nm-thick sections with a diamond knife on an ‘ultra-microtome’. We can look at cells or tissues.
Sections are collected onto copper grids (3mm across) and stained with lead or uranium salts. They are imaged using a ‘flooding’ beam of electrons (up to 120kV). The electrons produce light on a scintillator and the resultant photons are collected using a CCD camera. The image produced is actually the ‘electron shadow’ produced by the heavy metals in the sample and from the stain. We can obtain very high resolution from this technique, down to around 1nm.
The secret of good TEM is to fix and infiltrate samples very well. Unfortunately the use of glutaraldehyde in the fix means antigenicity is lost and antibodies rarely recognise the sections.
We offer a full sample preparation and sectioning service.
We can train you how to use the microscope or image your specimens for you.
We can also put semi-thin sections (650nm) on glass slides and stain them with toluidine blue; this produces a nicely preserved overview of the tissue which can be imaged on a light microscope.
In the very near future we hope to augment our new Jeol 1400 plus TEM with tomography so we will be able to make high-resolution, 3D reconstructions from slightly thicker (250 nm) sections.
SEM Scanning Electron microscopy
Exploring surfaces or subcellular structures of semi-thin larger specimens
SEM is usually performed on fixed, osmicated (treated with osmium tetroxide), completely dehydrated specimens. We can look at cells or tissues.
Specimens are mounted onto aluminium stubs in the preferred orientation. These are then coated with a layer of platinum or gold to make them conductive.
Our SEM system can be operated in different modalities using different detectors and specimen preparations, cell and tissues. They include the following:
- explore ultra-fine surface details with the secondary emission detector
- explore the structural detail of larger semi-thin sections akin to TEM images with the backscatter detector
- explore interior surfaces of cells and tissues using the resin section etching
SBF-SEM also known as 3-View
Using backscatter on serially exposed surfaces to create a 3D image
If you stain the sample with loads of heavy metals (lead salts, uranium salts and osmium twice!) it is possible to image the polished surface of a resin block with tissue in it even if you don’t make the whole sample conductive with a carbon coat.
With the in-built ultramicrotome of the SEM machine, you can cut a layer of sample away and image again... and again ... and again.
This allows you to generate a big Z-stack of images you can then analyse and ‘segment’ (i.e. pick out 3D structures). The limitations are: You can only look at very small blocks (0.65 x 0.6 x 0.5 mm).
You have to use a special detector for very low energy backscatter electrons (lowers resolution).
The samples are difficult to prepare and have a high failure rate. When it works though it is amazing!