In general, EELS is only applicable to thin TEM films, and EELS measurements of thicker films is possible only in a high-voltage TEM. In other words, the applicable thickness is proportional to the increase of mean-free-path length with the energy of the incident electron beam. On the other hand, another advantage of EELS in high voltage TEM is that characteristic angle θ_E, for inelastic scattering, decreases with increasing the energy of incident electron beam. [1]. Therefore, Thickness verification is important to ensure that the TEM sample is in the appropriate thickness range at the operating voltage.

[1] J. Śevely, J.Ph. P ́erez, B. Jouffrey: Energy loss of electrons through Al and carbon films from 300 keV up to 1200 keV, in [Ref. 1.78, p. 32]