Electron microscopy
Calibration of Magnification and Scale Bar in TEM
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In TEM measurements, the magnification of the image-forming lens system has normally an error of 5% - 10% so that the final magnification has an error of 5%-10%. To obtain more accurate magnification (and thus dimension measurement), e.g. in HRTEM, the magnification should be calibrated with a lattice image of a known standard specimen by recording both the specimen and standard (e.g. evaporated gold particles) in the same field of view. Furthermore, the instruments may give a few percent of image distortion which also needs to be calibrated.

For scale bar calibration in TEM and STEM modes, there are various calibration standards which can be obtained from EM accessory providers. However, the most common and accurate calibration methods are:

        i) At low magnifications, magnification calibration diffraction grating replica is commonly used. For instance, Ted Pella sells "500 nm" carbon replica with Au/Pd shadowing on a cross line grating with well-defined trench type grooves on square mesh copper grids. However, you will need to follow the instruction on your specimen vial to calculate the mesh size of your specific standard. To keep the calibration accuracy, at least 10 lines are needed in the field of view. At magnifications higher than that, the number of lines in the view become fewer, and thus it gets less precise as inconsistencies in the gratings become clearer.

It is very important to read the manual of the grating replica specimen and take care of it:
        i.a) Keep the replica in the vial when it is not in use.
        i.b) Do not touch the replica surface, clean or bend the replica specimen to avoid damage.
        i.c) To stabilize the specimen, sometimes a low illumination level is needed.

        ii) At medium magnifications, a TEM specimen prepared from a structure with good TEM or STEM contrast can be used for the scale bar calibration based on the calibration at low magnifications described in i). In this way, the medium scale bar calibration is transferred from the calibration obtained at low magnifications.

        iii) At high magnifications, especially for high resolution TEM, a holey carbon film shadowed with gold particles or a silicon crystal can be employed for calibration. In this case, lattice fringes are used.

In electron diffraction measurements, it is necessary to take a diffraction image from a known standard for calibration after obtaining the pattern of an unknown under the same electrical and magnetic conditions, specifically the same lens current and high voltage, and the same sample position. The known standard should have defined diffraction spacings (e.g. ring patterns induced by aluminum (Al) particles at very small crystallite size).