TEM Imaging Techniques
In TEM the beam illuminates the whole area of interest simultaneously and a grey-scale projection of the bulk thickness of the specimen is created.
This type of imaging is used for negatively stained particulate samples as well as sectioned specimens prepared for ultrastructure or immunodetection (see Specimen Preparation Techniques). It provides a relatively fast way to examine the specimen in 2D. Ultrathin sections of 50-200 µm or particles are mounted on specimen grids and imaged at nanometer resolution in x and y direction. The beam illuminates the whole area of interest simultaneously and a grey-scale projection of the bulk thickness of the specimen is created, limiting the resolution in the Z direction. Thicker areas as well as areas with higher concentration of heavy metals applied during preparation such as osmicated membranes in the kidney micorvili (left) or uranyl surrounding the negatively stained virus-like particles (right) appear darker than the background. The specimen grids can be stored and re-imaged multiple times.
Three-dimensional information is necessary for a good understaning of structure-function relationships in a biological specimen. A classical TEM image provides a 2D projection of the bulk of information contained in a 70 nm specimen section. Therefore the section thickness constitutes a resolution limit in the Z direction. In contrast to this, electron tomography allows acquisition of a number of images from one sample section at incremental angles and reconstruction of organelles in 3D. After processing and reconstruction, the final resolution is much better than the 70 nm limit of a traditional TEM section (5-20 nm).
Sample preparation follows the standard protocol. However, a thicker section of typically 200-300 nm is cut to provide more volume. Information from several serial sections can also be combined (serial tomography). During imaging the holder is tilted around its axis in 0.5-2° increments.
Our Talos L120C with LaB6 filament and Ceta camera is suited for screening of single particle samples in cryo modality. The Gatan 626.6 single specimen cryo-holder allows the user to screen approximately 4 grids per day, with a 40 minute holder warmup cycle between each grid. The user can choose to acquire the images using the microscope software or EPU.
The mode of imaging typically used for cryogen electron microscopy, but also applicable to other delicate specimens, is called low dose imaging. A cryospecimen is typically damaged after it receives a total of approximately 50 electrons per Å2. For 1s acquisition exposures at the recommended doses of <40 e-/Å2/s which allow sufficient contrast , this gives the user no more than a couple of seconds before the specimen melts and means that each point of the specimen can only be imaged once.
In low dose imaging the sample is first browsed at a low magnification and at a very low dose of <0.5e-/Å2, and therefore using a very noisy image, just good enough to find potentially good areas of ice (A in the figure below). To see specimen details, for example distribution of particles in the support film openings, we acquire images of a very limited area which is irradiated by the beam only during acquisition (C in the figure below). The beam is then automatically blanked using the pre-specimen shutter and condenser deflectors. The focusing and beam alignments are performed on a neighbouring area of support film (B in the figure below). The beam diameter is typically kept no larger than 2 µm to avoid damaging surrounding areas. The specimen is therefore either irradiated with extremely low dose at low magnifications (when browsing), or with a very small beam diameter at low dose (<40e-/Å2/s) during acquisition. The beam size, dose settings and beam offset for focusing between holes are stored separately by the software as search, focus and acquisition settings.
Apoferritin particles imaged on our Talos L120C.